Gemini Biosciences Limited

Extracellular Vesicles (EVs) are membrane-enclosed vesicles that can carry lipids, proteins, and RNAs, from one cell to another, for short or long distances. Depending on their size and origin, EVs can be characterized as exosomes (50–150nm in diameter), microvesicles (100nm–1μm in diameter) or large oncosomes (>1μm in diameter). Like Trojan horses, EVs can cross the cell barrier and reprogram cellular functions.

Gemini Biosciences offers a comprehensive and modular extracellular vesicles (exosomes) analysis service that involves production, purification and biochemical and biophysical analyses of the vesicles. Analysis techniques used include Nanoparticles Tracking Analysis (NTA) with the Nanosight NS300, fluorescence & electron microscopy, Western blotting, mass spectrometry (proteomics) and RNAseq.
We also offer a custom service involving the production, purification, and analysis of EVs from different cell lines subjected to various treatments according to the customer’s requirements. We have access to many human cancer and other cell lines available for use in customer experiments.
Please contact us for more information on cell lines available.

We have developed an efficient workflow for the purification and characterization of extracellular vesicles from biological fluids such as:

• Cell culture supernatants
• Serum
• Plasma
• Urine
• Cerebrospinal Fluid (CSF), etc.

We work with sample volumes from about 500μl – 10ml although this could be scaled up depending on the client's requirements. Our service is very flexible and we would be happy to discuss your project and how our service can support your project.

Analysis techniques we use include Nanoparticles Tracking Analysis (NTA), fluorescence & electron microscopy, Western blotting, mass spectrometry (proteomics) and RNAseq.

KEY ANALYSIS INSTRUMENTATION INCLUDES:

• Nanosight NS300 (Malvern) for Nanoparticles Tracking Analysis (NTA),
• Transmission electron microscope (JEOL JEM1400-Plus (120kV, LaB6) equipped with a Gatan OneView 4K camera
• Fluorescence microscope
• LTQ-Orbitrap mass spectrometer coupled to a Dionex NCP-3200 nanoLC system

Gemini Biosciences offers label-free quantitative proteomics analysis of customer samples where it is not convenient to metabolically or chemically label samples prior to analysis.

Label-free methods can provide a higher dynamic range of quantification, which means that we can measure significant changes within a complex mixture or across an entire proteome in a single experiment.

SILAC-based quantitative proteomics is an innovative service used in high throughput quantitative analysis of large protein complexes, chemical biology, biomarker discovery and protein-protein interactions. SILAC is an acronym that stands for Stable Isotope Labeling with Amino acids of Cells in culture. This approach provides an unbiased strategy that can reveal how specifically either inhibitors, or other perturbations, affect the dynamic properties and/or cellular distributions of proteins. It can also be used as a sensitive and effective method to determine the specific interaction partners of proteins under study. Although the technology is currently being used in basic investigative research mainly on continuously growing cell lines, we have developed strategies and procedures for using SILAC on differentiated cells.

SILAC-based quantitative proteomics is an innovative service used in high throughput quantitative analysis of large protein complexes, chemical biology, biomarker discovery and protein-protein interactions. SILAC is an acronym that stands for Stable Isotope Labeling with Amino acids of Cells in culture. This approach provides an unbiased strategy that can reveal how specifically either inhibitors, or other perturbations, affect the dynamic properties and/or cellular distributions of proteins. It can also be used as a sensitive and effective method to determine the specific interaction partners of proteins under study. Although the technology is currently being used in basic investigative research mainly on continuously growing cell lines, we have developed strategies and procedures for using SILAC on differentiated cells.

SILAC studies are performed using cells with the normal isotopes 12C and 14N in which the amino acids arginine and lysine are to be replaced with the heavy isotopes 13C and/or 15N. Heavy isotope substitution is made on the basic amino acids arginine and lysine because these are the sites of trypsin cleavage, thereby conveniently generating a set of tryptic peptides each with increased mass that can readily be detected and quantitated by mass spectrometry. When mammalian cells are grown using SILAC media, the proteins become substituted with the heavy forms of arginine and lysine. Subsequent MS/MS and software analyses resolves and quantitates the relative ratios of each isotopic form of every peptide. This measures whether the level of each protein in the complex has increased, decreased or remained at the same level after the treatment used. The accuracy of the quantitation is enhanced because the measurements are made at the level of individual peptides, while typically multiple (2-10+) peptides are analyzed for each protein. In this way the response of large numbers of endogenous, untagged proteins (many hundreds to thousands) in the cell or a specific complex can be evaluated quantitatively at multiple time points in a single experiment.

Gemini Biosciences offers custom SILAC tissue culture media and proteomics analysis service that includes advice on SILAC experimental design, sample processing, MS/MS analysis, data analysis and interpretation of results.

We offer a comprehensive phosphoproteomics service to our clients that includes experimental design, advice with sample preparation, fractionation, affinity enrichment of phosphopeptides using TiO2, phospho Serine/Threonine or phospho Tyrosine antibodies, LC-MS/MS, data analysis and interpretation. A final project report can be prepared at completion of each project according to the customer's needs.

Our quantitative phosphoproteomics analysis could either be done using metabolically labeled samples (using SILAC media), chemically labeled samples (iTRAQ) or a label-free quantitative proteomics approach depending on the nature of the customers samples.

Please contact us as early as possible during the planning stages of your project so that we can discuss the best approach that meets the requirements of your project.

METHYLATION PROTEOMICS

Gemini Biosciences methylation proteomics service allows the customer to obtain information on the methylation status of their biological samples. Our workflow involves using mono-methylated arginine specific antibodies to enrich for modified peptides in protease digested cell or tissue extracts. The antibody-purified modified peptides are then analysed by LC-MS/MS to determine modified sites and quantification of peptides/proteins to obtain quantitative profiles of hundreds of modified proteins in the customer's samples.

ACETYLATION PROTEOMICS

Our acetylation proteomics service provides the customer with information on the acethylation status of their biological samples. Our workflow involves using high affinity acetylated-lysine (Ac-K) specific antibodies to enrich for acetyl-modified peptides in protease digested cell or tissue extracts. The antibody-purified modified peptides are then analysed by LC-MS/MS to determine modified sites and quantification of peptides/proteins to obtain quantitative profiles of hundreds of modified proteins in the customer's samples.

UBIQUITINATION PROTEOMICS

Ubiquitin modification of cellular proteins is one of the major regulatory steps that directs protein degradation through regulation of proteasome activity. Since in most instances ubiquitination are the primary event in target selection, the system of ubiquitination and deubiquitination might be of similar importance. Furthermore, protein ubiquitination is involved in functions different from targeting proteins for degradation. Therefore, depending on the nature of the modification i.e. mono or polyubiquitination and on how ubiquitin chains are linked together, post-translational modifications of cellular proteins by covalent attachment of ubiquitin and ubiquitin-like proteins are involved in transcriptional regulation, receptor internalization, DNA repair, stabilization of protein complexes and autophagy.

The ubiquitin proteomics service offered by Gemini Bio uses the K-GG motif specific antibody to enrich for ubiquitinated substrates by pulling down ubiquitinated peptides in protease digested cell or tissue extracts. The antibody-purified modified peptides are then analysed by LC-MS/MS to determine modified sites and quantification of peptides/proteins to obtain quantitative profiles of the modified proteins in the customer's samples.

Monoclonal antibodies like polyclonal antibodies are widely used in life sciences research to identify proteins and study their cellular functions. They represent a single B lymphocyte generating antibodies to one specific epitope. B-cells can be isolated easily from the spleen and lymph nodes of immunised animals; however, these cells have a limited life span, and can only divide a limited number of times, coined the 'Hayflick limit'. This prohibits the culture of B-cells directly. For an antibody to be useful in research or industry, it must be readily available in large quantities. Due to the Hayflick limit, this would not be possible using B-cells cultured in vitro as they would eventually stop dividing and the population would die out.

Gemini Biosciences Monoclonal Antibody Package Options:

• Antibody production using customer supplied antigens (peptides, protein or other non-protein antigen
• Peptide design, synthesis and antibody production
• Protein expression/production and use of antigen for antibody production

Species Options:

• Mouse

Antigen Options:

• Recombinant protein
• Synthetic peptides (free antigen design and we can synthesize the antigen at a high purity for antibody production; see our peptide synthesis service

Purification Options:

• Protein A/G
• Fractional precipitation

If you are interested in developing a monoclonal antibody to your antigen of interest, please contact us for a quote and details on our antibody development process.

Polyclonal antibodies are widely used in biochemical and other biosciences applications to determine the cellular localisation and functions of proteins.

The immune response to an antigen generally involves the activation of multiple B-cells all of which target a specific epitope on that antigen. As a result a large number of antibodies are produced with different specificities and epitope affinities and these are known as polyclonal antibodies.

For production purposes these antibodies are generally purified from the serum of immunised animals where the antigen of interest stimulates the B-lymphocytes to produce a diverse range of immunoglobulin's specific to that antigen.

At Gemini Biosciences we produce high titre, high affinity polyclonal antibodies. These polyclonal antibodies can be used for research purposes in many areas of biology, such as immunoprecipitation, histochemistry, enzyme linked immunosorbent assays (ELISA), diagnosis of disease, immunoturbidimetric methods, western blots and Biochip technology. Polyclonal antibodies are ideally suited for use in sandwich assays as second stage antigen detectors.

Polyclonal Antibody Package Options:

• Antibody production using customer supplied antigens (peptides, protein or other non-protein antigen
• Peptide design, synthesis and antibody production
• Protein expression/production and use of antigen for antibody production

Species Options:

• Rabbit
• Sheep
• Mouse
• Rat
• Guinea Pig

Antigen Options:

• Recombinant protein
• Synthetic peptides (free antigen design and we can synthesize the antigen at a high purity for antibody production; see our peptide synthesis service)

Purification Options:

• Protein A /G.
• Cognate peptide antigen conjugated to activated beads (we can synthesize the antigen for antibody production and purification; see our peptide synthesis service).

Custom synthetic peptides are an important tool in life sciences research, drug discovery and proteomics. Gemini Bio provides a comprehensive peptide synthesis service to help customers in applications requiring custom antibody production, biochemical assays, peptide libraries and peptide arrays.

Gemini Biosciences offers a confidential and efficient peptide synthesis service at competitive prices.

Synthetic peptide applications

• Epitope-specific antibody production
• Study of protein functions and the identification and characterization of proteins
• Receptor binding or the substrate recognition specificity studies
• Peptide drugs
• Standards and reagents in mass spectrometry-based applications

Peptide modifications:

A comprehensive range of N-terminal, C-terminal and side chain modifications including: biotin, fluorescent labelling, phosphopeptides, stable isotopes and peptides for antibody production are available from Gemini Biosciences. We are experienced in the synthesis of difficult and hydrophobic peptide sequences and unusual peptide modifications.

Extensive range of purities and quantities

Peptides are available in milligram to gram quantities. We offer a range of peptide purities from crude peptides to >98% purity. Peptides are routinely synthesised from two up to sixty amino acids in length. However, depending on the customer's sequence, we can synthesize longer peptides.

Please contact us to discuss your peptide synthesis requirements and a quote.

During peptide array/library construction, 96 different peptides are synthesized in 96-well format. Each plate is individually tested for accuracy and can be used for epitope mapping, libraries, protein characterization and much more. At Gemini Biosciences we offer to customers up to 20-mer peptides at 1-5 mg scale. We also offer 3 tiers of analysis and delivery is only 2-3 weeks.

96 crude peptides per plate in 96-well format:

• 1- 3 mg scale (higher scale is available for additional charge, email us for price).
• N-terminal modification is available for addition charge (email us for price).
• Up to 20 amino acids in length.
• Guaranteed above 95% successful rate.
• For different format such as 48 well or 112, please contact us.
• No cross contamination.
• 2-3 week delivery.
• Statistically significant QC of each individual plate.

Gemini Biosciences offers three packages for peptide array/library construction i.e.

• Package 1: Peptide array synthesis with mass spectra for five statistically significant peptides
• Package 2: Synthesis with mass spectra for every peptide
• Package 3: Synthesis with mass spectrum and HPLC for all peptides

Please contact us to discuss your peptide synthesis requirements and a quote.

Gemini Biosciences' gene to protein service is a comprehensive and modular offer that includes gene synthesis, DNA cloning in customer’s preferred expression system and includes expression and purification of recombinant proteins according to the customer’s requirements.

Please contact us to discuss your gene to protein project and for a competitive quote.

Gene synthesis has become a powerful tool offering new solutions for molecular cloning, creation of fusion proteins and codon optimizations for achieving sufficiently high protein expression levels. Gemini Bio offers a gene synthesis service that is customisable to meet customer needs and with considerable savings of time and money compared to traditional molecular cloning using recombinant DNA technology.

Gemini Biosciences' gene synthesis service includes:

• 100% accuracy of synthesis guaranteed
• Genes up to 1kb are normally synthesised and shipped out in 2-3 weeks.
• Cloned into our standard vector or your vector of choice for a competitive price
• QC documentation by sequence chromatogram
• Free codon optimisation

The staff at Gemini Biosciences are experienced, PhD-level trained, and will ensure that you receive a high quality professional service tailored to your needs and delivery on schedule.

Send us the sequence of your gene of interest and we will do the rest according to your requirements.

As part of the gene synthesis and DNA cloning service, Gemini Biosciences offers site-directed mutagenesis that allows the customer to decide on any mutations they require in the sequence of their gene of interest.

Please contact us to discuss your project if you wish to introduce mutations in your gene of interest.

In life sciences and the pharmceutical industry, stable cell lines are used in many research applications including antibody production, recombinant protein production, assay development, gene editing, functional studies and more.

Gemini Biosciences strives to produce high quality stable cell lines using the customer's system of choice.

Gemini Biosciences can produce recombinant proteins to customers’ specifications from vectors supplied by them or produced by us as part of the DNA cloning service.

The general workflow includes:

• Transformation or transfection of cells with expression construct e.g. plasmid DNA or virus construct
• Identification and selection of over-expressing colonies or stable cell lines
• Small-scale expression and purification and assessment using SDS-PAGE
• Optimization of protein expression parameters
• Western blot (anti-tag) analysis of expressed protein if yield is low
• Estimation of protein yield
• Scale up growth of over-expressing strain s
• Cell harvesting and lysis
• Purification of protein from cleared lysate via purification tag or customer's preferred system
• SDS-PAGE or Western blot analysis of purified protein using anti expression tag antibody
• Determination of protein concentration
• Delivery of purified protein to customer

When required by the client, we can produce the recombinant protein on a large-scale following establishment of protocols.

If you are interested in generating recombinant proteins for any purpose, please contact us for details of our process and a quote.

Gemini Biosciences offers both a mass spectrometry (LC-MS/MS) and NMR-based metabolomics approach with high quality reproducible results. One aspect of the service involves metabolite profiling and identification from various matrices using high resolution mass spectrometry techniques.

When required by the client, the metabolomics analysis can include the quantitative analysis of changes in complex small molecule metabolite mixtures in biological systems to identify mechanisms associated with responses to stimuli which could be chemical, physical or biological.

Applications of metabolomics analyses include:

• Biomarker identification
• Monitoring of drug metabolism and toxicity (including during clinical trials and post-approval surveillance
• Metabolic profiling of in vivo and in vitro biological systems.

Gemini Biosciences uses the mass spectrometry (LC-MS/MS) or nuclear magnetic resonance (NMR) - based metabolomics approach to analyse client samples. The approach used in any study will depend on the client's requirements and experimental strategy that will provide the best results for their project. We can perform untargeted or targeted metabolomics analyses on customer samples based on their needs.

Critical information on the formation of metabolites including, where appropriate, both Phase I and Phase II metabolism and comparison of drug metabolism routes in different species is available.

Metabolomics studies carried out by Gemini Biosciences are undertaken as 'collaborative endeavours' and include support with experimental design, sample preparation, data acquisition, data processing and statistical analysis according to the client's requirements. We believe that only through collaboration and communication throughout a metabolomics or metabolite identification/profiling study can the greatest success of the project be obtained.

Please contact us to discuss your project and requirements and for details of our process and a quote.

Solution state NMR is the most versatile analytical tool for the biophysical analysis of proteins.

Solution-state NMR can be used for the following main purposes:

• determination of high- and atomic-resolution three-dimensional structures of proteins of molecular mass smaller than about 30-35kD.
• quick assessment of whether a recombinant protein is folded or unfolded, or in a molten globular state
• determination of whether and how a ligand binds to a protein. The ligand can be an inhibitor, natural ligand, cofactor, etc. The ligands can be small molecules, peptides, nucleic acids, carbohydrates, etc.
• determination of dissociation constants of weak binding ligands
• as initial screening of compounds and fragments in a drug discovery campaign
• metabolite characterisation.

Gemini Biosciences' NMR service includes the following:

• Designing appropriate approaches and workflow for a structural biology project
• Expression and production of pure Escherichia coli expressed recombinant protein in milligram quantities
• Production of uniformly and selectively 13C, 15N, 2H- labelled proteins and peptides
• Acquisition of multi-dimensional NMR data for structure calculations
• Determination of protein structures by NMR and X-ray crystallography
• Determining the structures of proteins and ligands using a combinationof NMR and computer docking.
• Sample preparation of tissues, cell extracts and biofluids for metabolomics studies.

The following hardware is available for use to deliver our customers' projects:

• 800 MHz spectrometer with high sensitivity cryogenic heads for solution studies

These three spectrometers are state-of-the-art and capable of performing most of the experiments that are essential for modern structural biology

Gemini Biosciences offers a high quality modular X-ray crystallography service to both academic and commercial clients at competitive rates.

X-ray crystallography is the leading technique for the structural elucidation of proteins and allows very high resolution three-dimensionl structures of biomolecules to be determined.

Crucial to this process is producing diffracting crystals and this is best done using a nano-drop crystallization robotic platform which will enable many crystallisation conditions to be rapidly screened. Among the challenging systems will be the crystallisation of membrane proteins and protein complexes.

A hotel system is also available for automated visualization and database archiving of crystal growth results.

Multi-Angle Light Scattering (MALS) provides an absolute, non-destructive technique for measuring basic solution properties of proteins. This method which is used by Gemini Biosciences is important for the following types of experiments:

• determination of the molecular mass and size of proteins,
• assessment of sample dispersity and aggregation
• prepare homogeneous, monodisperse proteins for NMR and X-ray crystallography.
• observation of molecular interactions in a real-time environment
• elucidation of conformation changes
• probing properties of partially folded proteins (precursors to protein aggregation/fibrillization)

Microcal ITC200 isothermal titration calorimeter used by Gemini Biosciences is the ‘gold standard’ for determining binding affinities.

The method will give information on the thermodynamics of binding, and the association constants. It is a method that is suitable for studying protein interactions with other proteins, peptides, nucleic acids and small molecules. The ITC method makes crucial contributions in the drug design process.

iTRAQ is an isobaric labeling method used in quantitative proteomics by tandem mass spectrometry to determine the amount of proteins from different sources in a single experiment. It uses stable isotope labeled molecules that can be covalent bonded to the N-terminus and side chain amines of proteins.

Where the customer’s project requires covalent chemical labeling of peptides prior to analysis, we offer iTRAQ as the approach for the quantitative proteomics analysis of experimental samples.

Our state of the art mass spectrometry equipment can be used to identify sites of phosphorylation from a protein in a 1D band or 2D gel spot. The 1D band or 2D spot must have good intensity by colloidal Coomassie staining in order to proceed; this gives us a better chance of identifying the sites of phosphorylation as the level of protein usually gives no indication of the stoichiometry of the phosphorylation present. Samples are normally processed and then in-gel digested with a protease e.g. trypsin, chymotrysin, etc. The digested samples are then extracted and are taken through three stages of analysis.

The stages include:

a) Identification of protein(s) by high-resolution mass spectrometry
b) Detection of phosphorylated peptides
c) Data interpretation and final reporting

Mascot reports of protein identification(s) and identification of sites of phosphorylation are provided electronically or as a hardcopy if requested.

The DNA cloning service at Gemini Biosciences is integrated with gene synthesis to facilitate the cloning of difficult genes into the customers’ vectors of choice.

The cloning service includes:

• Preparation of constructs from customers’ plasmids
• Production of expression constructs;
• Complete sequencing of cloned gene
• Detailed datasheet for completed constructs

Please contact us to discuss your project and obtain a competitive quote.

Gemini Biosciences offers a comprehensive gene to antibody production service that includes the following if the customer sends us the sequence of their gene of interest:

• Cloning of customer’s gene in an appropriate vector for expression of recombinant antigen
• Expression and purification of suitable mount of recombinant antigen in appropriate host
• Development of antibody in host species using recombinant antigen

Please contact us to discuss your gene to antibody project.

Bioparticle Technology Services