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Creative Proteomics

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Shirley, New York, US

About Creative Proteomics

Type: Privately Held Size: 51-200 employees

Creative Proteomics provides a comprehensive range of proteomics services using both gel-based and gel-free proteome analysis platforms. We have integrated a set of protein separation, characterization, identification and quantification systems into our proteomics workstation, which is featured with high... Show more »

Creative Proteomics provides a comprehensive range of proteomics services using both gel-based and gel-free proteome analysis platforms. We have integrated a set of protein separation, characterization, identification and quantification systems into our proteomics workstation, which is featured with high throughput and super-sensitivity. We offer quality proteomics services for target identification, lead optimization and preclinical and clinical phases of drug development.

Of note, Creative Proteomics is staffed by specialists who have extensive experience in handling hard-to-analyze samples, such as plasma membrane, serum, cerebrospinal fluid. In addition, we are also professional in studying protein modification and protein-protein interaction. Moreover, a series of proprietary proteomics data analysis programmers are available. In close cooperation with our partners, professional proteomics solutions are provided at the lowest cost in the industry.

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Our Services (61)


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Targeted Metabolomics

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Non-Targeted Metabolomics

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Peptide Synthesis

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Customized Synthesized Peptide/Proteins

Peptide Synthesis Service
In the quantitative proteomics research, several MS-based methodologies for relative quantification have been introduced for comparison of different proteomes from collected biological samples. Meanwhile, MS-based methods for absolute quantification of specific... Show more »

Customized Synthesized Peptide/Proteins

Peptide Synthesis Service
In the quantitative proteomics research, several MS-based methodologies for relative quantification have been introduced for comparison of different proteomes from collected biological samples. Meanwhile, MS-based methods for absolute quantification of specific proteins have been developed to accurately determine the protein concentrations. According to the guidelines for bioanalytical methods, the establishment and validation of accurate analytic proposals require standard compounds of high purity for calibrating and quality controls. Currently the dominant quantitative strategy is usually a combination of shotgun method and isotope dilution strategy. The targeted proteins in the complicated biological samples would release free peptide fragments induced by specific enzymatic cleavage, and the stable peptides with unique primary sequences in the digest mixture would be utilized as surrogates for corresponding parent proteins, so the small-molecular peptides can be quantified to estimate the protein concentration.

Scientist’s at Creative Proteomics specialized in the custom synthesis of synthetic peptides and peptide based molecules, providing a confidential and efficient service at competitive prices.

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Small Molecule Bioanalysis

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Founded & developing with the boom of medical Research & Development, Creative Proteomics is a young but competent CRO with experienced analysts & biochemists, focusing in bioanalysis of drug candidates, small molecules or macromolecules (protein and peptides) the customers required.

Equipped with advanced LC-MS instruments,... Show more »

Founded & developing with the boom of medical Research & Development, Creative Proteomics is a young but competent CRO with experienced analysts & biochemists, focusing in bioanalysis of drug candidates, small molecules or macromolecules (protein and peptides) the customers required.

Equipped with advanced LC-MS instruments, Creative Proteomics can establish and validate analytical methods for absolute quantification of small-molecule drugs & metabolites, or targeted proteins in various biological samples, according to GLP, and regulation agencies.

Creative Proteomics can provide professional bioanalytical service to all the clients around the globe. No matter you are from pharma giants, biotech companies, or academic labs, we can prepare robust, accurate & rapid analytical reports for your commissioned work.

Including:

Bioanalysis of Small Molecules
Bioanalysis of Proteins
ADME & PK
Preclinical Trials in Drug R & D
Bioanalysis of DNA Methylations
Residual DNA Testing
Ames Test
Identification of Bacterial Strains

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LC-MS/MS

Liquid chromatography tandem mass spectrometry
Price on request

LC-MS MRM Quantification

The protein quantification is based on isotope dilution LC-MS/MS and Multiple Reaction Monitoring (MRM) of specific peptides from the protein(s) of interest. The high sensitivity and specificity of LC-MS/MS method can be used for selective quantification of specific proteins by quantitating signature... Show more »

LC-MS MRM Quantification

The protein quantification is based on isotope dilution LC-MS/MS and Multiple Reaction Monitoring (MRM) of specific peptides from the protein(s) of interest. The high sensitivity and specificity of LC-MS/MS method can be used for selective quantification of specific proteins by quantitating signature peptides, in complex biological samples. This technique provides direct identification and quantification of the specific analyte and is complementary to RT-PCR, Western blotting and ELISA; combined with internal standards, the LC-MRM quantification shows high reproducibility (5% CV); and multiple targeted peptides/proteins can be identified and quantified in a single LC-MS run. About the protein/peptide standards for calibration, AQUA peptides, QconCATmer or PSAQ can be utilized, just as you require.

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Glycosylation Analysis

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A biosimilar, also known as follow-on biologic or subsequent entry biologic, is a biologic medical product which is a modified copy of an original product. Biosimilars are officially approved versions of original products, and only allowed to manufacture when the original one's patent expires.

Different from the small-molecule... Show more »

A biosimilar, also known as follow-on biologic or subsequent entry biologic, is a biologic medical product which is a modified copy of an original product. Biosimilars are officially approved versions of original products, and only allowed to manufacture when the original one's patent expires.

Different from the small-molecule drugs, macromolecular biologics generally exhibit high molecular complexity, and may be sensitive to tiny changes in manufacturing processes. Medical regulation authorities such as European Medicines Agency (EMA), Food and Drug Administration (FDA), and Health Canada, hold their own guidance for demonstration of the similarity of two biological products in terms of safety and efficacy. Like the me-too & me-better drugs, preclinical and clinical studies need to be performed to demonstrate safety, purity, and potency compared with reference biologics. But before the animal and clinical trials, the protein biologics themselves need to be characterized according to the guidance document ICH Q6B, which provides a uniform set of internationally accepted specifications for the characterization of biotechnological and biological products for approval.

Although the ICH Q6B document itself does not recommend any specific test procedure or specific acceptance criteria, it do suggest technical approaches, to provide the following information for biotechnological or biological products:

1 Structural Characterization
Amino Acid Sequence
Amino Acid Composition
N/C-terminal Sequencing
Peptide Mapping
Disulfide bond Analysis
Glycosylation Analysis

2 Physicochemical Properties
Molecular Weight(MW) & Size
Isoform Pattern
Extinction Coefficient(EC)
Electrophoretic Patterns
Liquid Chromatographic Patterns
Spectroscopic Profiles

3 Process- & Product-related Impurities
Host Cell Proteins
Truncated Forms
Post-translational Modifications(PTM)
Aggregates

Creative Proteomics is able to provides a full range of analytical services mentioned above, to characterize your biopharmaceutical products. All the analytical proposals are designed and performed in a GMP compliant laboratory using various validated techniques for regulatory requirements.

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MALDI Tissue Imaging

Matrix-Assisted Laser Desorption Ionization Tissue Imaging
Price on request
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Lipidomics

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Lipids are hydrophobic or amphipathic small molecules which include fats, waxes, sterols, fat-soluble vitamins (such as vitamins A, D, E and K), monoglycerides, diglycerides and phospholipids. The crucial role of lipids in biological physiology is evident not only in energy storage and structural components of cellular membranes,... Show more »

Lipids are hydrophobic or amphipathic small molecules which include fats, waxes, sterols, fat-soluble vitamins (such as vitamins A, D, E and K), monoglycerides, diglycerides and phospholipids. The crucial role of lipids in biological physiology is evident not only in energy storage and structural components of cellular membranes, but also in signal transduction, membrane trafficking and morphogenesis.

Currently many modern technologies, including mass spectrometry (MS), nuclear magnetic resonance (NMR), fluorescence spectroscopy, column chromatography, and microfluidic devices are utilized to identify, quantify, and understand the structure and function of lipids in biological systems.

Mass spectrometry has proved highly efficient for the characterization and quantification of lipid species in thelipid extracts, leading to unparalleled selectivity, sensitivity and the ability to provide structural information for components in complex mixtures.

Equipped with advanced tandem mass spectrometry for determination of all 8 sorts of lipid and powerful bioinformatic software packages, Creative Proteomics has a panel of experienced scientists and technicians, and provide customer-tailored service with rapid analysis procedures and easy to read report, to speed your research and publications.

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Metabolomics

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Metabolomics is the study of metabolism, specifically the science of identifying and quantifying the biochemical byproducts of metabolism, called cellular metabolites. This is achievedby using analytical technologies such as NMR and mass spectrometry combined with sophisticated statistical methods to interpret the generated data.... Show more »

Metabolomics is the study of metabolism, specifically the science of identifying and quantifying the biochemical byproducts of metabolism, called cellular metabolites. This is achievedby using analytical technologies such as NMR and mass spectrometry combined with sophisticated statistical methods to interpret the generated data. Compared with genomics, transcriptomics and proteomics, metabolomics provide a direct and global snapshot of all the metabolites, and tell the researchers what have happened, making it more and more popular in disease research, toxicology, environmental analysis, agriculture, biofuel development and nutrition.

The routine procedure of metabolomic research can be divided into untargeted and targeted. The formeraims for a quick and reliable identification of small molecule metabolites for a particular physiological state in response to internal or external perturbations; and the latter provides an accurate quantitation of metabolites the researcher are interested in, able to screen and validate biomarkers for medical research.

Creative Proteomics is a professional and component CRO with experienced bioanalysts and technicians, which can provide reliable and customer-tailored service, with rapid analytical procedures to speed up your research, and detailed and easy-to-read report for your publications.

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Protein Arrays

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Protein Microarray, a powerful tool, contains hundreds of molecules, including cytokines, phophatases, receptors and other proteins at one plate. Scientists from Creative-Proteomics offer you the high-density microarray with thousands of unique, full length proteins, which can be detected at the same time, to facilitate your research.

Protein Microarray, a powerful tool, contains hundreds of molecules, including cytokines, phophatases, receptors and other proteins at one plate. Scientists from Creative-Proteomics offer you the high-density microarray with thousands of unique, full length proteins, which can be detected at the same time, to facilitate your research.

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Protein Characterization

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The application of Bottom-Up strategy is limited by incomplete or ambiguous characterization of alternative splice forms, diverse modifications and endogenous protein cleavages. As an alternative, the Top-Down strategy has been utilized more with the advancement of analytical instruments. Except for FT-ICR mass spectrometer,... Show more »

The application of Bottom-Up strategy is limited by incomplete or ambiguous characterization of alternative splice forms, diverse modifications and endogenous protein cleavages. As an alternative, the Top-Down strategy has been utilized more with the advancement of analytical instruments. Except for FT-ICR mass spectrometer, invention and innovation of orbitrap mass spectrometer has promote the development of top-down proteomics.

Because there is no digestion of proteins before analysis, the information which is lost when shotgun method is used, for example, the quaternary structure with disulfide bonds, can be maintained and detected when the Top-Down strategy is applied, which show unique advantage in protein-level characterization of disulfide bonds. This make it get more and more application in QC analysis of protein therapeutics, such as insulin, to make the recombinant protein are correctly expressed, folded and assembled.

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Protein Structure Determination

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The application of Bottom-Up strategy is limited by incomplete or ambiguous characterization of alternative splice forms, diverse modifications and endogenous protein cleavages. As an alternative, the Top-Down strategy has been utilized more with the advancement of analytical instruments. Except for FT-ICR mass spectrometer,... Show more »

The application of Bottom-Up strategy is limited by incomplete or ambiguous characterization of alternative splice forms, diverse modifications and endogenous protein cleavages. As an alternative, the Top-Down strategy has been utilized more with the advancement of analytical instruments. Except for FT-ICR mass spectrometer, invention and innovation of orbitrap mass spectrometer has promote the development of top-down proteomics.

Because there is no digestion of proteins before analysis, the information which is lost when shotgun method is used, for example, the quaternary structure with disulfide bonds, can be maintained and detected when the Top-Down strategy is applied, which show unique advantage in protein-level characterization of disulfide bonds. This make it get more and more application in QC analysis of protein therapeutics, such as insulin, to make the recombinant protein are correctly expressed, folded and assembled.

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In vitro ADME/DMPK Studies

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Pharmacokinetics quantitatively how the body affects a specific drug after administration through the mechanisms of absorption and distribution, as well as the chemical changes of the substance in the body (e.g. by metabolic enzymes such as cytochrome P450 or glucuronosyltransferase enzymes), and the effects and routes of... Show more »

Pharmacokinetics quantitatively how the body affects a specific drug after administration through the mechanisms of absorption and distribution, as well as the chemical changes of the substance in the body (e.g. by metabolic enzymes such as cytochrome P450 or glucuronosyltransferase enzymes), and the effects and routes of excretion of the metabolites of the drug. Usually 4 steps are inovlved in the pharmacokinetics procedure:

Absorption: the process of a substance entering the blood circulation;
Distribution: the dispersion/dissemination of substances throughout the body fluids, tissues and organs;
Metabolization: the recognition and irreversible transformation of parent compounds into daughter metabolites by metabolic enzymes;
Excretion: the removal of the substances from the body.

The two phases of metabolism and excretion can also be grouped together under the title elimination. The metabolites, Phase I or Phase II, maybe activated drugs (if the original compound is pre-drug), less toxic, or even more poisonous compounds, also need to be identified and quantified. The parameters would be directly measured and calculated using professional bioinformatics software WinNonlin, which is widely accepted by the academic and pharmaceutical industry. And in order to obtain a quantitative description of the ADME procedure, the parameters below are usually utilized:

Cmax The peak plasma concentration of a drug after administration;
tmax Time to reach Cmax;
Cmin The lowest (trough) concentration that a drug reaches before the next dose is administered;
Vd Volume of distribution
T1/2 Elimination half-life
ke Elimination rate constant
AUC Area under the curve
CL Clearance
f Bioavailability
Creative Proteomics is equipped with stable LC-MS systems, and staffed with experienced analysts and technicians, who are familar with the analytical work-flow and requirements from the regulation agenicies. Although the significance of automation is emphasized in bioanalytical procedures, Creative Proteomics always insist that the dedication and the skill of the staff are essential for success. So any specific question you may have can get optimal advices and rapid solution from our experienced an professional techical team.

http://www.creative-proteomics.com/application/adme-and-pk.htm

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Biomolecular Interaction Analysis

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Surface Plasmon Resonance, SPR for short, is defined as the excitation of surface plasmon (collective charge oscillation along the interface between two materials) by incident light under the condition of total internal reflection. During SPR, the surface electrons of the interface resonate at the same frequency as the light. But... Show more »

Surface Plasmon Resonance, SPR for short, is defined as the excitation of surface plasmon (collective charge oscillation along the interface between two materials) by incident light under the condition of total internal reflection. During SPR, the surface electrons of the interface resonate at the same frequency as the light. But the adsorption of biomolecules onto the interface would change oscillation frequency of surface electrons. Several SPR-based biosensors, such as auto-lab, Biacore and ProteOn XPR36 have been developed for application in biochemical researches, and the most popular technique based on SPR, is Biacore for accurate characterization of biomolecular interaction.

Currently the Biacore technique, an optical-based detection system, is considered as an ideal tool to explore biomolecules interactions of various pairs of partners. When the the analytes in the solution passing through bind with the immobilized ligands on the surface, Biacore can detect the changes of reflection angle close to the metal surface. Immobilizing different ligands on various sensor surface (the most popular one is CM5), allows the monitoring of the following biomolecular interactions:

Protein-Protein Interaction

Nucleic Acid-Protein Interaction

Protein-Small Molecule Interaction

With the Biacore technique, Creative Proteomics can perform real-time monitoring of the binding/dissociation of analytes & corresponding ligands, which provides rapid stoichiometric kinetic, affinity and thermodynamic measurement. The advantages above, make Biacore popular in:

Immunologic Test & Biochemical Research

Drug Discovery & Screening

Nucleic Acid-Nucleic Acid & Nucleic Acid-Protein Interaction

Proteomics Research

With advanced Biacore instrument, various sensor chips and experienced technical staff, Creative Proteomics can provide cost-effective characterization service of biomolecular interactions.

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Protein Aggregation Analysis

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Compared with small molecule drugs, recombinant protein therapeutics require more strict control in bio-synthesis, purification and storage steps. Given the instability & degradation, there is the high tendency of macromolecules to aggregate under the processing & storage conditions. The aggregation of protein therapeutics is the... Show more »

Compared with small molecule drugs, recombinant protein therapeutics require more strict control in bio-synthesis, purification and storage steps. Given the instability & degradation, there is the high tendency of macromolecules to aggregate under the processing & storage conditions. The aggregation of protein therapeutics is the most common and troubling manifestation during R & D, because protein aggregates usually exhibit reduced or no biological activities since the medical biologics are functional only in native state; more importantly, they might show stronger immunogenicity/cellular toxicities. So the protein aggregation must be controlled strictly to a satisfactorily low level before commercialized.

Size Exclusion HPLC (SE-HPLC) has become an essential analytical tool for the detection and analysis of soluble protein aggregates. The separation mechanism is based on the protein shape and size. Proteins of different sizes elute at different rates through the column; the larger the protein particles are, the earlier they are eluted. In Creative Proteomics, the analytical lab is equipped with Size Exclusion HPLC system for this services above.

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Peptide Mapping

Price on request

It is important to make sure the protein therapeutics, such as recombinant proteins, peptides and monoclonal antibodies, are biosynthesized correctly. Peptide mapping works as a powerful analytical approach to confirm the identity of a specific protein, detail characterization of the protein& screen and identify PTMs. The purified... Show more »

It is important to make sure the protein therapeutics, such as recombinant proteins, peptides and monoclonal antibodies, are biosynthesized correctly. Peptide mapping works as a powerful analytical approach to confirm the identity of a specific protein, detail characterization of the protein& screen and identify PTMs. The purified proteins/peptides are reduced into short peptides by protease cleavage or chemical cleavage, then separated and analyzed with MALDI-TOF and ESI-TOF o identify the peptide sequence by peptide mass fingerprints. A cocktail of proteases usually are used to generate more unique peptides.

Our technical team of scientists pioneered the optimal methods used for peptide and protein sequencing. When peptide mapping alone is not enough, Creative Proteomics can utilize a range of techniques, LC-MS, LC-MS/MS, Nanospray to ensure your protein is sequenced to the extent possible.

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Host Cell Protein (HCP) Detection

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Because most of recombinant proteins are synthesized by cell-based systems in biochemical researches, the host cells derived from bacteria, yeast, mammalian cells, insect cells and plants (such as rice and tobacco) can be used for protein therapeutics manufacturing. But limited by current purification techniques, low levels (1 to... Show more »

Because most of recombinant proteins are synthesized by cell-based systems in biochemical researches, the host cells derived from bacteria, yeast, mammalian cells, insect cells and plants (such as rice and tobacco) can be used for protein therapeutics manufacturing. But limited by current purification techniques, low levels (1 to 100 ppm) of host cell proteins (HCPs) may still remain in the purified biotherapeutics, even after a series of purifications. The ppm-level contaminants in biotherapeutics may trigger an unpredictable immune response in patients after dosing, and are required to be identified and quantified as part of drug safety evaluation, by the regulatory agencies.

Usually the HCP assay can be achieved by 2D Electrophoresis, WesternBlot and ELISA. After efficient separation of the biotherapeutics with electrophoresis, the host cell proteins would be identified and quantified with antiserum or polyclonal antibodies. LC-MS/MS, as a powerful analytical technique, has been introduced for HCP profile as an attractive supplement to immunoassays.

In Creative Proteomics, the tech panel can provide 3 analytical procedures:

1D PAGE (IEF or SDS) and HCP Identification by MS;

2D PAGE and HCP Identification by MS;

LC-MS/MS analysis for HCPs

The protein therapeutics are separated with 1D/ 2D electrophoresis, and then visualized by Coomassie brilliant blue or sliver staining, and then digested into peptides by in-gel digestion for mass spectrometry analysis. Meanwhile, the protein therapeutics can be digested directly for nanoLC-ESI-LTQ/Orbitrap system. The contaminant proteins can be detected by database searching. Although PAGE has certain limitations, such as in pI, MW range, and protein hydrophobicity, but it can help to detect specific proteins if not fractionationed with electrophoresis. Creative Proteomics can help you to select the better one.

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Amino Acid Analysis

Price on request

If an exact knowledge of protein/peptide quantities is required for further applications, quantitative amino acid analysis, qAAA, is a suitable assay, which can not only determine protein quantities precisely, but also provide detailed information regarding the relative amino acid composition and free amino acids. The AAA... Show more »

If an exact knowledge of protein/peptide quantities is required for further applications, quantitative amino acid analysis, qAAA, is a suitable assay, which can not only determine protein quantities precisely, but also provide detailed information regarding the relative amino acid composition and free amino acids. The AAA procedure includes hydrolysis, separation, detection and quantification.

Hydrolysis is typically achieved under acid conditions. A standard procedure is hydrolysis with 6 M hydrochloric acid (24 hours, 110°C). Fragile amino acids, especially tryptophan and cysteine, will be partially destroyed. And then hydrolyzed samples (amino acids) are derivatized pre-column or post column for sensitive detection, separated by RP/SCX column. The use of internal and external standards of known amount is crucial for accurate quantification of each amino acid.

Equipped with automatic amino acid analyzer, Creative Proteomics can provide highly reproducible quantitative analysis of protein/peptide samples the customers submit, for estimation of amino acid composition of specific proteins, and purity determination.

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DNA Pull Down Assay

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Pull-Down assay is an in vitro method used to determine a physical interaction between proteins. It can be used to confirm a protein-protein interaction discovered through other techniques, and to explore previously unknown protein-protein interactions of specific proteins.

Pull-Down assay is quite similar to... Show more »

Pull-Down assay is an in vitro method used to determine a physical interaction between proteins. It can be used to confirm a protein-protein interaction discovered through other techniques, and to explore previously unknown protein-protein interactions of specific proteins.

Pull-Down assay is quite similar to immunoprecipitation(IP), except that a bait protein replaces an antibody as affinity reagent. Pull-Down assay can greatly enhance the speed and efficiency of protein purification and exploration of potential binding partners, especially the ones stably bound to bait protein . In a Pull-Down assay, the bait proteins are usually tagged with GST, polyHis, or biotin, and captured on an immobilized affinity ligand against the tags, such as glutathione nickel column or streptavidin, and thereby generating a "secondary affinity support"' for isolating proteins bound to bait proteins. The secondary affinity support of immobilized bait is then incubated within a biological sample, to capture putative prey proteins. And then protein complex would be washed to eliminate unspecific binding. Identification of bait-prey interactions requires that the complex is removed from the affinity support and analyzed by detection methods, such as SDS-PAGE. But SDS-PAGE loading buffer is harsh which will denature all proteins in the sample. And competitive analyte elution is much more specific for the bait-prey interaction.

Besides confirmation and discovery of interactions between proteins, Pull-Down assay is also a powerful tool to detect the activation status of specific proteins. For example, proteins activated in response to tyrosine phosphorylation, can be pulled down with an immobilized SH2 domain which targets the phosphorylated tyrosine on a given protein. And this method is highly specific to detect whether distinct proteins are activated. The tech team of Creative Proteomics can have provide professional analytical support to the customers after having a detailed conversation with you.

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Protein-Protein Interaction Analysis

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Proteins are the critical players in molecular recognition at the core of all biological processes. They can interact with the other components of cells, such as small-molecule metabolites, nucleic acids, membranes, and other proteins to build supramolecular assemblies and elaborate molecular machines that perform all sorts of... Show more »

Proteins are the critical players in molecular recognition at the core of all biological processes. They can interact with the other components of cells, such as small-molecule metabolites, nucleic acids, membranes, and other proteins to build supramolecular assemblies and elaborate molecular machines that perform all sorts of functions, from chemical catalysis and mechanical work to signaling and regulation. As one of the most important interactions, Protein-Protein Interactions have been studied widely. So far large scale protein-protein interactions have been identified, and all the generated data collected together in specialized databases, enables the creation of large protein interaction networks.

Like the metabolic or genetic/epigenetic networks, the research of PPIs can help us to understand the mechanism of signal transduction, transportation across membranes, cell metabolism and other biological processes conducted by stable or transient, covalent or non-covalent interactions.

Information stored in PPIs databases such as DIP (Database of Interacting Proteins), BIND(Biomolecular Interaction Network Database) and KEGG (Kyoto Encyclopedia of Genes and Genomes), can be applied to establish protein-protein interactions networks. With the in-house software, Creative Proteomics can help you to establish and visualize a more reliable PPI networks by scoring the confidence level of putative interactions.

Creative Proteomics can provide even senior data mining services, using protein annotation information, such as:

1 Prediction of interacting sites of proteins;

2 Reliability Assessment of Protein-Protein Interactions;

3 Establishment of interaction networks for specific collections of proteins, and involved with pathological/physiological processing.

Establishment of interaction networks for specific collections of proteins, and involved with pathological/physiological processing.

Co-immunoprecipitation (co-IP)
Pull-Down Assay
Crosslinking Protein Interaction Analysis
Label Transfer Protein Interaction Analysis
Far-Western Blot Analysis

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iTRAQ Protein Labeling

Isobaric Tags for Relative and Absolute Quantitation
Starting at $100.00 per sample

Creative Proteomics offers iTRAQ protein quantification service suited for unbiased untargeted biomarker discovery. Relative quantification of proteins for biomarker discovery in complex mixtures by mass spectrometry can easily and quickly be achieved using iTRAQ technology. iTRAQ is ideally suited for comparing normal, diseased,... Show more »

Creative Proteomics offers iTRAQ protein quantification service suited for unbiased untargeted biomarker discovery. Relative quantification of proteins for biomarker discovery in complex mixtures by mass spectrometry can easily and quickly be achieved using iTRAQ technology. iTRAQ is ideally suited for comparing normal, diseased, and drug-treated samples, time course studies, biological replicates and provides relative quantitation.

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DNA Methylation Analysis

Price on request

The transfer of one-carbon methyl groups to nitrogen or oxygen (N- and O-methylation, respectively) to amino acid side chains increases the hydrophobicity of the protein and can neutralize a negative amino acid charge when bound to carboxylic acids. Methylation is mediated by methyltransferases, and S-adenosyl methionine (SAM) is... Show more »

The transfer of one-carbon methyl groups to nitrogen or oxygen (N- and O-methylation, respectively) to amino acid side chains increases the hydrophobicity of the protein and can neutralize a negative amino acid charge when bound to carboxylic acids. Methylation is mediated by methyltransferases, and S-adenosyl methionine (SAM) is the primary methyl group donor. Methylation occurs so often that SAM has been suggested to be the most-used substrate in enzymatic reactions afterATP. Additionally, while N-methylation is irreversible, O-methylation is potentially reversible.

Methylation is a well-known mechanism of epigenetic regulation, as histone methylation and demethylation influences the availability of DNA for transcription. Amino acid residues can be conjugated to a single methyl group or multiple methyl groups to increase the effects of modification.

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Ubiquitylation Analysis

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Ubiquitin is an about 8.5 kDa polypeptide consisting of 76 amino acids that is appended to the ε-NH2 of lysine in target proteins via the C-terminal glycine of ubiquitin. Following an initial monoubiquitination event, the formation of a ubiquitin polymer may occur, and polyubiquitinated proteins are then recognized by the 26S... Show more »

Ubiquitin is an about 8.5 kDa polypeptide consisting of 76 amino acids that is appended to the ε-NH2 of lysine in target proteins via the C-terminal glycine of ubiquitin. Following an initial monoubiquitination event, the formation of a ubiquitin polymer may occur, and polyubiquitinated proteins are then recognized by the 26S proteasome that catalyzes the degradation of the ubiquitinated protein and the recycling of ubiquitin.

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Phosphorylation Analysis

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Protein phosphorylation is the most commonly studied area of post-translational modification since it plays a vital role in intracellular signal transduction and is involved in regulating cell cycle progression, differentiation, transformation, development, peptide hormone response, and adaptation. It has been estimated that one... Show more »

Protein phosphorylation is the most commonly studied area of post-translational modification since it plays a vital role in intracellular signal transduction and is involved in regulating cell cycle progression, differentiation, transformation, development, peptide hormone response, and adaptation. It has been estimated that one third of mammalian proteins may be phosphorylated and this modification often plays a key role in modulating protein function. Reversible protein phosphorylation, principally on serine, threonine or tyrosine residues, is one of the most important and well-studied post-translational modifications.

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Label Free Quantitative Proteomics

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Creative Proteomics provides Label-free methods for both relative and absolute quantification, which a rapid and low-cost alternative to other quantitative proteomic approaches.

Label-free quantification is a method in mass spectrometry that aims to determine the relative amount of proteins in two or more biological samples.... Show more »

Creative Proteomics provides Label-free methods for both relative and absolute quantification, which a rapid and low-cost alternative to other quantitative proteomic approaches.

Label-free quantification is a method in mass spectrometry that aims to determine the relative amount of proteins in two or more biological samples. Unlike other methods for protein quantification, label-free quantification does not use a stable isotope containing compound to chemically bind to and thus label the protein.

Label-free quantification may be based on precursor signal intensity or on spectral counting. The computational framework of label free approach includes detecting peptides, matching the corresponding peptides across multiple LC-MS data, selecting discriminatory peptides. The first method is useful when applied to high precision mass spectra, such as those obtained using the new generation of time-of-flight (ToF), fourier transform ion cyclotron resonance (FTICR), or Orbitrap mass analyzers. The high-resolution power facilitates the extraction of peptide signals on the MS1 level and thus uncouples the quantification from the identification process. In contrast, spectral counting simply counts the number of spectra identified for a given peptide in different biological samples and then integrates the results for all measured peptides of the protein(s) that are quantified.

Typically, peptide signals are detected at the MS1 level and distinguished from chemical noise through their characteristic isotopic pattern. These patterns are then tracked across the retention time dimension and used to reconstruct a chromatographic elution profile of the mono-isotopic peptide mass. The total ion current of the peptide signal is then integrated and used as a quantitative measurement of the original peptide concentration. For each detected peptide, all isotopic peaks are first found and the charge state is then assigned.

In contrast to differential labeling, every biological specimen needs to be measured separately in a label-free experiment. The extracted peptide signals are then mapped across few or multiple LC-MS measurements using their coordinates on the mass-to-charge and retention-time dimensions. Data from high mass precision instruments greatly facilitate this process and increase the certainty of matching correct peptide signals across runs.

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Post-Translational Modification Analysis

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Creative Proteomics offers an advanced analytical platform for the characterization of various post-translational modifications(PTM). As a significant approach to increase proteomic diversity, PTMs play a key role in many cellular processes such as cellular differentiation, protein degradation, signaling and regulatory processes,... Show more »

Creative Proteomics offers an advanced analytical platform for the characterization of various post-translational modifications(PTM). As a significant approach to increase proteomic diversity, PTMs play a key role in many cellular processes such as cellular differentiation, protein degradation, signaling and regulatory processes, regulation of gene expression, and protein-protein interactions. These modifications include phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, acetylation, lipidation and proteolysis and influence almost all aspects of normal cell biology and pathogenesis. Protein post-translational modification (PTM) increases the functional diversity of the proteome by the covalent addition of functional groups, such as phosphate, acetate, amide groups, or methyl groups, to specific proteins, and influence almost all the aspects of normal cell biology and pathogenesis. Some of them are listed below:

glycosylation, addition of a glycosyl group to either asparagine, hydroxylysine, serine, or threonine;
acetylation, the addition of an acetyl group, usually at the N-terminus of the protein;
alkylation, the addition of an alkyl group;
methylation, the addition of a methyl group, usually at lysine or arginine residues;
biotinylation, acylation of conserved lysine residues with a biotin appendage;
glutamylation, covalent linkage of glutamic acid residues to tubulin and some other proteins;
glycylation, covalent linkage of one to more than 40 glycine residues to the tubulin C-terminal tail of the amino acid sequence.

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Protein AQUA

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Absolute Quantification is a targeted quantitative proteomics technique that exhibits robust efficacy and is being increasingly utilized for a wide variety of quantitative proteomics studies. AQUA strategy is for the absolute quantification (AQUA) of proteins and their modification states. Peptides are synthesized with... Show more »

Absolute Quantification is a targeted quantitative proteomics technique that exhibits robust efficacy and is being increasingly utilized for a wide variety of quantitative proteomics studies. AQUA strategy is for the absolute quantification (AQUA) of proteins and their modification states. Peptides are synthesized with incorporated stable isotopes as ideal internal standards to mimic native peptides formed by proteolysis. These synthetic peptides can also be prepared with covalent modifications (e. g. , phosphorylation, methylation, acetylation, etc.) that are chemically identical to naturally occurring posttranslational modifications. Such AQUA internal standard peptides are then used to precisely and quantitatively measure the absolute levels of proteins and post-translationally modified proteins after proteolysis by using a selected reaction monitoring analysis in a tandem mass spectrometer.

Advances in biological mass spectrometry have resulted in the development of numerous strategies for the large-scale quantification of protein expression levels within cells. Besides the measurements of protein expression accomplished through differential incorporation of stable isotopes into cellular proteins, the absolute quantification is a useful method in proteomics analysis.

The absolute quantification strategy: a general procedure for the quantification of proteins and post-translational modification. AQUA provides absolute quantification by employing synthetic peptides containing stable isotopes.

The absolute quantification method is based on the discovery of an unexpected relationship between MS signal response and protein concentration: the average MS signal response for the three most intense tryptic peptides per mole of protein is constant within a coefficient of variation of less than 10%. Given an internal standard, this relationship is used to calculate a universal signal response factor. The universal signal response factor (counts/mol) was shown to be the same for all proteins tested.

While isotope methods establish only relative quantification of expressed proteins, the absolute quantification (AQUA) strategy can provide information for the precise determination of protein expression and post-translational modification levels. The AQUA method relies on the use of a synthetic internal standard peptide that is introduced at a known concentration to cell lysates during digestion. Analysis of the proteolyzed sample by a selected reaction monitoring (SRM) experiment in a tandem mass spectrometer results in the direct detection and quantification of both the native peptide and isotope labeled AQUA internal standard peptide.

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DIGE

Differential gel electrophoresis
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Creative Proteomics provides DIGE service which allows for simultaneous separation of up to three samples on one gel, bringing a new level of statistical confidence and reliability to 2D gel electrophoresis.

Differential in Gel Electrophoresis (DIGE) is a technique to monitor the differences in proteomic profile between cells... Show more »

Creative Proteomics provides DIGE service which allows for simultaneous separation of up to three samples on one gel, bringing a new level of statistical confidence and reliability to 2D gel electrophoresis.

Differential in Gel Electrophoresis (DIGE) is a technique to monitor the differences in proteomic profile between cells in different functional states. This is done in three steps. First, samples are tagged with unique fluorescent dyes. Second, they are run together on the same 2D-PAGE gel. Finally, after the run completes, the different fluorescent images of the same gel are superimposed over each other. DIGE allows the study of proteins that are expressed differentially, as well as those that are common between samples. This technology allows for simultaneous separation and comparison of up to three samples on one gel.

In traditional 2D-PAGE, different samples are often separated in multiple gels. Those gels are then overlapped to compare one gel to another. Due to differences between gels in spatial resolution and spot intensities, the overlaying of images and correct matching of proteins is difficult. There can also be variations in protein uptake by the isoelectric focusing strips, incomplete protein transfer from the first to the second dimension gel, and local inconsistencies in gel composition, field strength or pH gradients. These gel-to-gel variations can mask the biological variation between the samples. Unfortunately, not all of these variations are avoidable; they can occur for a number of reasons. Thus, quantitative comparisons of protein expression levels are difficult using 2D-PAGE.

In DIGE, protein mixtures are pre-labeled, prior to electrophoresis, with cyanine dyes that guarantee co-migration of proteins. This co-migration on the same gel eliminates running differences between samples. In addition, the samples are subjected to the same environment and the same procedures throughout the experiment. Experimental variation is minimized in this way.

The cyanine dyes (Cy2, Cy3 and Cy5) used for DIGE are N-hydroxy succinimidyl ester derivatives, are covalently tagged to the ε-amino group of lysine residue of proteins, and replace the ε-amino group positive charge with the positive charge of the dye. The binding of the dyes introduce small but matched increases in molecular weight to the protein. Since these dyes are hydrophobic, to prevent precipitation of proteins, only one lysine residue per protein is labeled (minimal labeling). Minimal labeling limits the fluorescence intensity and thus the sensitivity of the stain. The dyes are all charge-matched and molecular mass-matched to prevent alterations of the isoelectric point, and to minimize dye-induced shifting of labeled proteins during electrophoresis.

The cyanine dyes (Cy2, Cy3 and Cy5) used for DIGE are N-hydroxy succinimidyl ester derivatives, are covalently tagged to the ε-amino group of lysine residue of proteins, and replace the ε-amino group positive charge with the positive charge of the dye. The binding of the dyes introduce small but matched increases in molecular weight to the protein. Since these dyes are hydrophobic, to prevent precipitation of proteins, only one lysine residue per protein is labeled (minimal labeling). Minimal labeling limits the fluorescence intensity and thus the sensitivity of the stain. The dyes are all charge-matched and molecular mass-matched to prevent alterations of the isoelectric point, and to minimize dye-induced shifting of labeled proteins during electrophoresis.

To study differential expression of proteins, there is no need to run multiple gels. Loss of proteins even in the low molecular weight range is reduced since no post-electrophoretic processing (fixation or destaining) is necessary. This method is more quantitative than the standard colorimetric staining methods, both with regard to sensitivity as well as linearity.

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Peptide Mass Fingerprinting

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Creative Proteomics provides Peptide Mass Fingerprinting (PMF) analysis and ions searching against database for rapid identification of proteins.

Peptide mass fingerprinting (PMF) is an analytical technique for protein identification. Basically, the unknown protein of interest is first cleaved into smaller peptides, whose... Show more »

Creative Proteomics provides Peptide Mass Fingerprinting (PMF) analysis and ions searching against database for rapid identification of proteins.

Peptide mass fingerprinting (PMF) is an analytical technique for protein identification. Basically, the unknown protein of interest is first cleaved into smaller peptides, whose absolute masses can be accurately measured with a mass spectrometer such as MALDI-TOF or ESI-TOF. Then these masses are compared to either a database containing known protein sequences or even the genome sequence which can be translated into proteins through computer programs. Then the absolute masses of the peptides from each protein are calculated theoretically for mass comparison between the peptides of the unknown protein and the theoretical peptide masses of each protein to find the best match.

The advantage of PMF method is that only the masses of the peptides is need to be known, while time-consuming de novo peptide sequencing is then unnecessary, as long as the protein sequence is present in the database of interest. Additionally, most PMF algorithms assume that the peptides come from a single protein while the presence of a mixture can significantly complicate the analysis and potentially compromise the results, thus an isolated protein is needed for the PMF based protein identification. Mixtures exceeding a number of 2-3 proteins typically require the additional use of MS/MS based protein identification to achieve sufficient specificity of identification.

In sample preparation for PMF, protein samples can be derived from SDS-PAGE and then subject to some chemical modifications. Disulfide bridges in proteins are reduced and cysteine amino acids are carbamidomethylated chemically or acrylamidated during the gel electrophoresis. Then the proteins are cut into several fragments using proteolytic enzymes to generate peptides for mass spectrometric analysis.

The digested protein can be analyzed with different types of mass spectrometers such as ESI-TOF or MALDI-TOF. MALDI-TOF is often the preferred instrument because it allows a high sample throughput and several proteins can be analyzed in a single experiment, if complemented by MS/MS analysis.

A small fraction of the peptide is pipetted onto a MALDI target and a chemical called a matrix and then the matrix and peptide molecules co-crystallize on the MALDI target and are ready to be analyzed. The target is inserted into the vacuum chamber of the mass spectrometer and the desorption and ionisation of the polypeptide fragments is initiated by a pulsed laser beam which transfers high amounts of energy into the matrix molecules. The energy transfer is sufficient to promote the ionisation and transition of matrix molecules and peptides from the solid phase into the gas phase. The ions are accelerated in the electric field of the mass spectrometer and fly towards an ion detector where their arrival is detected as an electric signal. Their mass-to-charge ratio is proportional to their time of flight (TOF) in the drift tube and can be calculated accordingly. The mass spectrometric analysis produces a list of molecular weights of the fragments which is often called a peak list for computational analysis. The peptide masses are compared to protein databases such as Swissprot, NCBInr, which contain protein sequence information. The results are statistically analyzed and possible matches are returned.

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Protein Digestion

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Among the endoproteases which could be used for protein digestion, the serine protease trypsin is most commonly employed as it generates peptides which are highly amenable to MS(/MS) analysis. Depending on the preceding workflow, the enzymatic digestion of proteins is performed either in-gel or in-solution, generally, the in-gel... Show more »

Among the endoproteases which could be used for protein digestion, the serine protease trypsin is most commonly employed as it generates peptides which are highly amenable to MS(/MS) analysis. Depending on the preceding workflow, the enzymatic digestion of proteins is performed either in-gel or in-solution, generally, the in-gel digestion methodology has become routine for proteins separated by 2-D electrophoresis while in-solution digestion are usually used in LC-MS/MS analysis. The protein is cut enzymatically into a limited number of shorter fragments during digestion and these fragments are called peptides and allow for the identification of the protein with their characteristic mass and pattern.

In-Gel Digestion

Following the separation of samples by 1-D or 2-D gel electrophoresis, proteins are fixed and visualized using an MS-compatible stain, usually Coomassie Blue, or silver employing a glutaraldehyde-free protocol. Visualized proteins are excised from the gel and the respective gel bands or spots are washed for destaining and dehydrated before being trypsinized.

The permeation of the enzyme to the gel is believed to be facilitated by the dehydration of the gel pieces by treatment with acetonitrile and subsequent swelling in the digestion buffer containing the protease. Different studies about the penetration of the enzymes to the gel showed the process to be almost completely driven by diffusion, by cutting the gel to pieces as small as possible the efficiency of the in-gel digestion could be achieved.

Surfactant (detergents) can aid in the solubilization and denaturing of proteins in the gel and thereby shorten digestion times and increase protein cleavage and the number and amount of extracted peptides, especially for lipophilic proteins such as membrane proteins. Cleavable detergents are detergents that are cleaved after digestion, often under acidic conditions. This makes the addition of detergents compatible with mass spectrometry.

In order to ensure efficient hydrolysis of those proteins embedded in the gel matrix, a relatively high enzyme concentration is generally used. The generated proteolytic peptides can subsequently be released from the gel matrix, by for example 50% acetonitrile (ACN)/5% formic acid (FA) being used as an extraction buffer, in combination with sonication. To meet the requirements of peptides with different physical and chemical properties an iterative extraction with basic or acidic solutions is performed.

The efficiency of in-gel digestion and peptide extraction depends on a variety of factors, including: (i) the physico-chemical properties of the proteins and resulting peptides (e. g. , degree of hydrophobicity, size, amino acid sequence); (ii) the composition, size, and thickness of the gel pieces; (iii) the composition of the extraction buffer (e. g. , acetonitrile (ACN) concentration); (iv) the type of enzyme and its specific activity; (v) the general reaction conditions (e. g. , temperature, time, ratio of enzyme to substrate); (vi) the type of protein stain (e. g. , Coomassie or silver).

One significant advantage of in-gel protein digestion is that any contaminants (e. g. , detergents, salts) are already removed during electrophoresis, and that the generated peptide samples can be readily subjected to (LC/) ESI-MS analysis. However, the effectiveness of this procedure can be limited due to poor accessibility of the protease to proteins and/or inefficient release of peptides from the gel matrix, as well as inadequate storage of gels. As an excellent alternative to gel electrophoresis combined with in-gel digestion, proteins can be directly digested in-solution, which is usually followed by 1-D or 2-D LC to effectively separate the resulting peptide mixture before ESI-MS.

In-Solution Digestion

For in-solution digestion, a compromise between protein solubilization and enzyme activity must be reached. In the reduction and alkylation (r&a) of the cystines or cysteines potentially embodied in the protein, the disulfide bonds of the proteins are irreversibly broken up and the optimal unfolding of the tertiary structure is obtained. This chemical modification allows for proteins with a high number of disulfide bonds the successful identification as well as the highest peptide yield and sequence coverage. With denaturing electrophoresis it is strongly recommended to perform the reaction before the execution of the electrophoresis, since there are free acrylamide monomers in the gel able to modify cysteines.

Detergents should be generally avoided but, depending on the physico-chemical characteristics of the proteinlysate, organic solvents and/or chaotropes may be used. For the analysis of proteins which are difficult to solubilize or denature (e. g. , membrane proteins), 8M urea may be used in conjunction with the resilient protease Lys-C; subsequently, trypsinization is performed at a reduced concentration of chaotrope (2M urea). Following acidification using FA it may be possible directly to perform LC/ESI-MS, although it is generally recommended that an additional desalting step be performed. As a promising alternative to double proteolytic digestionin urea, organic solvents can be used to solubilize and effectively digest hydrophobic proteins in-solution. In order to enhance proteolytic digestion, enzymes that are chemically immobilized or physically adsorbed to a stationary phase can be used

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2D Gel Image Analysis

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The evaluation of the spot patterns is the most demanding part of the 2-D electrophoresis technique, and the images would be analyzed comparatively and automatically. 2-D gel patterns are compared to detect qualitative and/or quantitative protein expression changes between individual samples or different populations. Imaging... Show more »

The evaluation of the spot patterns is the most demanding part of the 2-D electrophoresis technique, and the images would be analyzed comparatively and automatically. 2-D gel patterns are compared to detect qualitative and/or quantitative protein expression changes between individual samples or different populations. Imaging analysis of 2-D gels can provide various types of information for the detection of novel, missing, or modified proteins; quantification of protein spots; determine of pI and Mr values of protein spots; enzyme digest and mass spectrometry detection.

For the evaluation and comparison of the complex 2-D patterns, the gel images have to be converted into digital data with a scanner or camera for further analysis with computer software. In imaging analysis, usually a pixel format of 100 mm is applied for scanning, higher pixel resolution does not improve imaging analysis and the image files will become too big to be processed in a reasonable time and take up a lot of space on the hard disk.

The scanner must be calibrated using a gray step tablet. The measured dimensionless intensity is converted into OD values or absorbance units. For accurate picking of spots according to the data of imaging analysis, the x/y positions need to be absolutely correct in the mm tolerance range. Usually the x/y data have to be calibrated for each scanner with the help of a grid. These calibration data have to be imported into the spot picker computer for each scanner used.

In imaging analysis of 2-D patterns, a background subtraction function can easily lead to errors. After many years of experience with spot volume quantification, it has been found that by defining the spot boundary at 75% of the peak maximum and calculating the spot volume for only the values above the boundary, the relative spot volume quantification comes very close to the real situation and ensures a very high reproducibility.

In normalization of the variation in protein loading and staining, the spot volumes are normalized against the total spot volume of the gel fully automatically without any user interaction.

As for calibration of pI and Mr Annotation, two ways are usually involved.

1-D calibration. Calibration curves are derived from IPG pH gradient graphs for pI determination, from the positions of co-run molecular weight markers for the second dimension Mr determination by importing a ladder.

2-D calibration. In 2-D calibration, pI and Mr values are calibrated at the same time by interpolating between known values which usually come from mass spectrometry analysis. A number of spots with known protein properties are selected, the pI and Mr Information are imported from the respective protein lists belonging to these spots.

For differential analysis it is important to be able to superimpose the spot patterns. This cannot be done directly, because typically there are local distortions caused by imperfections in gel matrix, variations in gel running conditions, temperature effects, uneven focusing, and polymerization issues. Formerly spot matching tried to find corresponding spots in pairs of gels, while for automatic matching it is more efficient to search for pairs of features, using spot clusters, spot shapes, sizes, and positions together. However, even this feature-based matching can sometimes produce mismatches, therefore it is necessary to control this process by visual inspection, and to perform corrections with setting landmarks.

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Western Blot

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The transfer or "blotting" of electrophoretic separated proteins from the gel matrix to a membrane (typically nitrocellulose or PVDF) followed by subsequent antibody-based detection on the surface of the membrane is called Western Blot or Immunoblot. Creative Proteomics provides western blotting analysis for the detection of a... Show more »

The transfer or "blotting" of electrophoretic separated proteins from the gel matrix to a membrane (typically nitrocellulose or PVDF) followed by subsequent antibody-based detection on the surface of the membrane is called Western Blot or Immunoblot. Creative Proteomics provides western blotting analysis for the detection of a specific target protein out of a complex protein mixture, e. g. tissue homogenate or cell extract, using highly selective and sensitive antibody-antigen interactions. The resulting data allow both qualitative and semi-quantitative analysis of the protein of interest.

The Western Blot is a widely used analytical technique for the detection of specific proteins in a sample of tissue homogenate or extract. It uses gel electrophoresis to separate native proteins by 3-D structure, or denatured proteins by the length of the polypeptide. The separated proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are stained with antibodies specific to the target protein. Western Blot is widely used in the fields of molecular biology, biochemistry, immune genetics and other molecular biology disciplines. Blotting is the transfer of macromolecules on immobilizing membranes for specific and sensitive detection.

In Western Blot analysis, the proteins of the sample are first separated in gel electrophoresis by isoelectric point (pI), molecular weight, electric charge, or a combination of these factors using various methods such as SDS-PAGE and IEF. Usually SDS-PAGE is employed for the separation, because all the proteins are solubilized and migrate in the same direction, and the epitopes are easier accessible due to the denaturing effect of SDS.

After the gel electrophoresis analysis, the proteins are moved from within the gel onto a membrane made of nitrocellulose or polyvinylidenedifluoride (PVDF) to make them accessible for antibody detection. PVDF membranes have a higher binding capacity for proteins than nitrocellulose, but nitrocellulose binds small proteins better. The primary method for transferring the proteins is called electroblotting which uses an electric current to pull proteins from the gel into the PVDF or nitrocellulose membrane. There are several ways to perform the electrophoretic transfer: tank blotting, semi-dry blotting and semi-wet blotting. All three have in common is that the gel and the membrane form a sandwich with a stack of filter papers on both sides. It should be noted that, if isoelectric focusing is used for the separation of the proteins, it is more efficient to transfer the proteins by diffusion with pressure blotting. During electroblotting, the proteins move from within the gel onto the membrane while maintaining the organization they had within the gel.

After the blotting process, which can take about an hour (semidry blotting) to overnight (tank blotting), the proteins are exposed on a thin surface layer for antibody detection. Besides, the free binding sites of the membrane are blocked with a protein mixture, which will not interfere with the subsequent probing with an antibody. The proteins are first detected by the primary antibody, and then detected with a secondary antibody, which recognizes this particular primary antibody. The secondary antibody is conjugated with a set of specific molecules, which can be easily detected with a subsequent development procedure with high sensitivity.

The most sensitive detection methods are using enhanced chemiluminescence (ECL): the antibody–horseradish conjugate recognizes the primary antibody; the substrate reaction is coupled with a secondary reaction which causes chemiluminescent light emission for a certain time period. This light signal is accumulated by exposing the membrane on an X-ray film, or by placing it into an absolutely dark cabinet where the signal is recorded with a sensitive CCD camera. With a special variant of ECL, down to 1 pg of a protein band is detectable.

Western Blotting could be used to follow protein phosphorylation. An antibody that binds to all the isoforms of a poly-phosphorylated protein will show a 'beads-on-a-string" look on a 2D gel. Western Blotting is also useful for identifying known and unknown proteins in complexes brought down by co-immunoprecipitation. Once proteins of interest are located on a Western blot, corresponding spots can be cut from a duplicate Coomassie blue-stained gel and identified by MS.

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PAGE

Polyacrylamide Gel Electrophoresis
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In biological membranes, many proteins are organized in complexes. Creative Proteomics offers a method for the global analysis of the subunits of these protein complexes through 2D Blue Native / SDS-PAGE analysis. In 2D BN / SDS-PAGE analysis, the samples were analyzed in the 1st dimension by blue native polyacrylamide gel... Show more »

In biological membranes, many proteins are organized in complexes. Creative Proteomics offers a method for the global analysis of the subunits of these protein complexes through 2D Blue Native / SDS-PAGE analysis. In 2D BN / SDS-PAGE analysis, the samples were analyzed in the 1st dimension by blue native polyacrylamide gel electrophoresis (BN-PAGE), and then separated by SDS-PAGE in the 2nd dimension which is 90 degrees from the first.

BN-PAGE provides the technology for high resolution separation of protein complexes. The blue native electophoresis protocol is used to determine the size, relative abundance and subunit composition of protein complexes. Protein complexes organize and maintain the cellular and organelle functions on all levels of complexity in time and space, including cell development and division, transcription and translation, respiration and photosynthesis, transport and metabolism. Protein complexes may exhibit partial and temporal changes, as well as exhibit different stabilities, making the identification and analysis extremely difficult.

In 2D BN/SDS-PAGE analysis, the protein or protein complex samples are separated under native conditions in a first-dimension BN-PAGE. The Coomassie blue dye, which is negatively charged and binds nonspecifically to all the proteins, is used in BN-PAGE. Coomassie blue does not act as a detergent, and it preserves the structure of protein complexes. In addition, the binding of Coomassie blue decreases the tendency of proteins to aggregate during the stacking step of the electrophoresis process. Therefore, the electrophoretic mobility of the samples is determined by the negative charge of the bound Coomassie blue and the size and shape of the complex.

For 2D BN/SDS-PAGE, the proteins and protein complex samples are denatured by SDS in the gel strip after the separation by BN-PAGE before they are applied to a second-dimension SDS-PAGE gel.

By combination of first dimension BN-PAGE and second dimension SDS-PAGE, monomeric proteins will migrate in a hyperbolic diagonal due to the gradient gel in the first and a linear gel in the second dimension, while components of protein complex are located below this diagonal. Proteins that represent subunits of the same protein complex can be found in one vertical line in the second dimension, whereas several spots of the same protein in a horizontal line indicate the presence of the protein in several distinct protein complexes.

2D BN/SDS-PAGE can provide information about the size, number, protein composition, stoichiometry and relative abundance of the sample, and has been proved to be a useful tool for the study of protein complex.

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SILAC

Stable Isotope Labeling by Amino Acids in Cell Culture
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SILAC-based quantitative proteomics (SILAQ) is an innovative technology used in high throughput quantitative analysis of large protein complexes, protein-protein and protein-small molecule interactions. SILAC service of Creative Proteomics provides an unbiased strategy that can reveal how specifically either inhibitors, or other... Show more »

SILAC-based quantitative proteomics (SILAQ) is an innovative technology used in high throughput quantitative analysis of large protein complexes, protein-protein and protein-small molecule interactions. SILAC service of Creative Proteomics provides an unbiased strategy that can reveal how specifically either inhibitors, or other perturbations, affect the dynamic properties and cellular distributions of proteins. It can also be used as a sensitive and effective method to determine the specific interaction partners of proteins in the cell.

Quantitative proteomics has increasingly gained impact in life science research as a tool to describe changes in protein expression between different cellular states. Stable isotope labeling by amino acids in cell culture (SILAC) is a powerful technique for relative quantification of proteins. Stable isotope labeling by amino acids in cell culture (SILAC) is a simple and straightforward approach for in vivo incorporation of a label into proteins for mass spectrometry (MS)-based quantitative proteomics. SILAC relies on metabolic incorporation of a given 'light' or 'heavy' form of the amino acid into the proteins. As the two isotopically labeled amino acids are essentially chemically identical, their incorporation does not interfere with normal cell growth, while leading to proteins/peptides that are distinguishable by mass and thus are ideal for mass spectrometric analysis. The method relies on the incorporation of amino acids with substituted stable isotopic nuclei. Thus in an experiment, two cell populations are grown in culture media that are identical except that one of them contains a 'light' and the other a 'heavy' form of a particular amino acid (e. g. 12C and 13C labeled L-lysine, respectively). When the labeled analog of an amino acid is supplied to cells in culture instead of the natural amino acid, it is incorporated into all newly synthesized proteins. After a number of cell divisions, each instance of this particular amino acid will be replaced by its isotope labeled analog. Since there is hardly any chemical difference between the labeled amino acid and the natural amino acid isotopes, the cells behave exactly like the control cell population grown in the presence of normal amino acid.

In comparation with derivatization-based labeling techniques directly after harvesting them for subsequent purification steps and analysis, the advantage of SILAC is that it ensures maximum reproducibility and minimum sample variation with regard to the protein level.

The SILAC samples are then subjected to enzymatic digestion and LC/MS analysis. The protein quantification is therefore carried out on the peptide level by comparing the peak the same amino acid composition and sequence but different masses. In addition to the determination of protein levels, SILAC approaches are well suited for monitoring changes in post-translational modifications. Examples for these applications include the measurement of changes in protein phosphorylation and methylation.

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Protein Quantification

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Absolute Quantification is a targeted quantitative proteomics technique that exhibits robust efficacy and is being increasingly utilized for a wide variety of quantitative proteomics studies. AQUA strategy is for the absolute quantification (AQUA) of proteins and their modification states. Peptides are synthesized with... Show more »

Absolute Quantification is a targeted quantitative proteomics technique that exhibits robust efficacy and is being increasingly utilized for a wide variety of quantitative proteomics studies. AQUA strategy is for the absolute quantification (AQUA) of proteins and their modification states. Peptides are synthesized with incorporated stable isotopes as ideal internal standards to mimic native peptides formed by proteolysis. These synthetic peptides can also be prepared with covalent modifications (e. g. , phosphorylation, methylation, acetylation, etc.) that are chemically identical to naturally occurring posttranslational modifications. Such AQUA internal standard peptides are then used to precisely and quantitatively measure the absolute levels of proteins and post-translationally modified proteins after proteolysis by using a selected reaction monitoring analysis in a tandem mass spectrometer.

Label-free quantification is a method in mass spectrometry that aims to determine the relative amount of proteins in two or more biological samples. Unlike other methods for protein quantification, label-free quantification does not use a stable isotope containing compound to chemically bind to and thus label the protein.

Label-free quantification may be based on precursor signal intensity or on spectral counting. The computational framework of label free approach includes detecting peptides, matching the corresponding peptides across multiple LC-MS data, selecting discriminatory peptides. The first method is useful when applied to high precision mass spectra, such as those obtained using the new generation of time-of-flight (ToF), fourier transform ion cyclotron resonance (FTICR), or Orbitrap mass analyzers. The high-resolution power facilitates the extraction of peptide signals on the MS1 level and thus uncouples the quantification from the identification process. In contrast, spectral counting simply counts the number of spectra identified for a given peptide in different biological samples and then integrates the results for all measured peptides of the protein(s) that are quantified.

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Spectroscopy

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Synthetic Chemistry

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Electrophoresis

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Epigenetic Studies

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Imaging & Spectroscopy

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Image Analysis

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Sequencing data in FASTQ

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Pharmacology & Toxicology

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Bioanalysis

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Separation/Purification Services

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Protein Sequencing

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Liquid Chromatography Coupled Mass Spectrometry Methods

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Protein Services

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Mass Spectrometry

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Omics

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Protein Expression Visualization

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Gel Electrophoresis

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