Alamo Laboratories Inc

Purified proteins/cell lysate/ tissue lysate samples will be run on SDS-polyacrylamide gels followed by transfer of the fractionated proteins/polypeptides/fragments onto PVDF membrane. The membrane is probed with a primary-secondary antibody pair via chemiluminescent method to detect the protein/fragment of interest. This service needs to be optimized on a case basis to suit the optimal fractionation of protein samples provided by the customer.

Time Line: 5-20 Days.

Price: $195/run (Price Valid till December 20, 2015)

Summary of Service: Western Blot Run (WB) will be optimized to fractionate the purified proteins/cell lysate/ tissue lysate samples on SDS-polyacrylamide gels followed by transfer of the fractionated proteins/polypeptides/fragments onto PVDF or nitrocellulose membrane. The membrane is probed with a primary-secondary antibody pair via chemiluminescent method to detect the protein/fragment of interest. If STD proteins are not transferred, we will repeat the electrophoresis, blotting and probing steps to obtain a satisfactory chemiluminescent signal. This service often needs to be optimized on a case basis to suit the optimal fractionation of protein samples provided by the customer.

Number of samples per run: 1-8

WB System: (1) Reducing or Non-Reducing SDS-gel (2) Linear Gradient (4-20% SDS-PAGE Gel) or Fixed conc. (4, 10, 12% or custom SDS-PAGE Gel) (3) TRIS-Glycine buffer system or any other system preferred by customer (4) Choice of marker/standard proteins (5) Wet-transfer of proteins on PVDF membrane (6) Probing of WB membrane with one primary - secondary antibody pair and visualization by chemiluminescent method.

Deliverables: Digital image of the blot via email.

Requirements: Customer provides the following

(1) Pure Protein samples in Sample Buffer (>50ug/protein/run; Conc. = >1mg/ml) or Cell/tissue lysates in Sample Buffer (>500ug/sample/run; Conc. = >1mg/ml). Alternatively, we may attempt to solublize the samples in Sample buffer for a nominal fee ($10/pure protein sample).
(2) Primary-secondary antibody pair to detect the protein/fragment of interest. Alternatively, customer may purchase the antibodies available from us.

Available cell lines and tissues:
- HeLa, MCF-7, A549, PC3, SK-N-SH, NIH/3T3,
LNCaP, SuDHL-16, CAPAN-1, Mosquito cell line
c6/36
- Mouse, Rat and Human organs/tissues.

Capabilities:
- Kit-based (provided by the researcher), custom designed ELISA
- Indirect, Sandwich, competitive
- Homogenization/Sonication of cell/tissue lysates
- Transfection/Transduction of mammalian/non-mammalian cells
- Optimization of protocols for commercial kits
- Analyze serum, plasma, cells, tissues
- Analyze chromogenic substrates

Equipment:
- Multi-channel and automatic pipets

Number of samples processed in a single experiment: 96-well plate format

Typical turn-around time: 14 days

ELISA with TMB substrate. Primary Antibody, standards or ELISA kit needs to be provided.

Sensitivity of the assay: 20 to 2000 micro-grams/ml of protein. We can run up to 25 Test Samples + 8 Standard samples (all in triplicates) either in 96 well plate or in micro-tubes. We will provide the researcher Standard Curve graph, equation for the regression analysis and generating the Standard curve (including values for slopes, intercept etc) and protein content of the unknown samples. Reagents: Our assay-report will include the source and catalog numbers of all materials used in the assay (including Standard Protein). Equipment used for the assay: Molecular Devices SpectraMax Plus, SpectraMax 250 or Cary UV-Vis Spectrophotometer. Turn around time: 1 to 10 days. Left-over samples: If researcher needs the remainder of the Unknown Samples back after the assay, the actual shipping charges will be added to the estimate. We also offer Lowry, MicroBCA, 660nm Protein Assay and CUSTOM protein assays for unknown samples containing common salts, detergents, reducing agents etc.

Sensitivity of the assay: 1 to 1500 micro-grams/ml of protein. S We can run up to 25 Test Samples + 8 Standard samples (all in triplicates) either in 96 well plate or in micro-tubes. We will provide the researcher Standard Curve graph, equation for the regression analysis and generating the Standard curve (including values for slopes, intercept etc) and protein content of the unknown samples. Reagents: Our assay-report will include the source and catalog numbers of all materials used in the assay (including Standard Protein). Equipment used for the assay: Molecular Devices SpectraMax Plus, SpectraMax 250 or Cary UV-Vis Spectrophotometer. Turn around time: 1 to 10 days. Left-over samples: If researcher needs the remainder of the Unknown Samples back after the assay, the actual shipping charges will be added to the estimate. We also offer Lowry, MicroBCA, 660nm Protein Assay and CUSTOM protein assays for unknown samples containing common salts, detergents, reducing agents etc.

Anti-Microtubule Compound Screening

Screening of the Drugs for the Anti-microtubule or Anti-tubulin Activity:
Drug-induced inhibition of the assembly of the mammalian microtubule protein or tubulin or tubulin isotypes is measured to assess the potency of the drug. From this test, one should be able to identify those drugs from a given library, which are active against microtubules, tubulin or one of its isoforms.
Microtubule Protein (5 mg/ml) or Tubulin Protein (1 mg/ml) will be polymerized in the presence/absence of the test-compound and the polymerization will be followed by turbidimetry (change in absorbance at 340 nm). Total amount of the polymers in the steady state (where microtubule polymers are in equilibrium with the free tubulin), formed in the presence or absence of the test- compound, will be compared. All the experiments will be run in duplicate.

Result-format for Tests:
(1) The ORIGINAL DA340 values of control samples (in the absence of the test- compound).
(2) The ORIGINAL DA340 values of test samples (in the presence of the test- compound).
(3) The percent inhibition/stimulation of microtubule polymerization by the test- compound.

Test 1: Sensitivity of the Microtubules to perspective drugs/compounds:
This basic screening utilizes drug-induced inhibition of the assembly of the mammalian MTs (mixture of tubulin and MAPs) to assess the potency of the drug. Unless specified, we will test the drugs at 1 and 100 mM concentrations. From this test, one should be able to identify those drugs from a given library, which are active against at least one of the constituents of microtubules (tubulin or one of the MAPs).
PRICE: $90 per compound

Test 2: Anti-tubulin (MAP-free) Activity of test- compounds:
Our high thorough-put screening utilizes the drug-induced inhibition of polymerization of the purified mammalian tubulin (MAP-free), to assess the potency of the lead compound. Unless specified, we will test the drugs at 1 and 100 mM concentrations. The results from this screening can resolve whether or not the anti-mitotic activity of the compounds will arise from their interaction with tubulin.
PRICE: $125 per compound

Test 3: Screening of the Drugs/Compounds for the Tubulin Isotype Specific Reactivity:
The screening at Tests 1 and 2 utilizes tubulin, which is a mixture of at least 7 isotypes, each of the a and b-tubulins. Depending on the choice of customers, we can test the effect of a compound on the assembly of all or one of the following isotypes: abII, abIII and abIV. Unless specified, we will test the drugs at 300 nM and 30 mM concentrations. From this test, one should be able to identify whether or not, a given drug can differentiate among the isotypes.
PRICE: $250 per compound

Test 4: Determination of IC50 of the Drugs for the Microtubule Assembly:
This service is an extension of the Tests-1 through 3. The inhibitory constant for the drug (s) will be determined using the tubulin polymerization assay. ALAMO LABS will test the effect of a compound (1 nM to 1 mM) on the assembly of either microtubule protein, pure tubulin or one of the isotypes of tubulin chosen by the customer. From this test, one should be able to appraise the potency of a given drug relative to other drugs in the same library or to those reported in literature.

Test 5: Determination of Binding Constant (Ka) of the Drug(s)/compounds for tubulin:
The apparent affinity constant (Ka) of the drug(s)/compound(s) for purified mammalian tubulin or its isotypes is measured. The interaction is measured at 20 different concentrations (1 nM to 30 mM) of the drug/compound. The Ka values, which are invariably related to the anti-microtubule potency of the drug (measured at Tests-1 through-5) will assist the customer to sort out the lead compounds/drugs from a library for in vivo evaluation of their anti-microtubule potency.

Test 6: Cell Toxicity of the drug(s):
Most of the anti-tubulin/anti-microtubule compounds inhibit the normal dynamic organization of the MTs that is necessary for the cell division. We can evaluate the effect of a given drug/compound on any cell line of customer’s choice, using a cell proliferation/ viability assay to appraise the anti-proliferative/viability potency of a given drug relative to the other drugs in the same library or to those reported in literature.

Custom Polyclonal Antibodies:
- Antigen Design
- Peptide Synthesis
- Polyclonal Antibody production
- Affinity Purification

Standard Peptide Anti-Sera Production in Rabbits
We take pride in Design and Synthesis of peptides and production of polyclonal antibodies in rabbits. Alamo® Labs offers a variety of Custom-Sera-production protocols and packages to suit the need of any researcher. Besides 70-day standard protocol, we also offer an Extended Protocol with 6-Immunizations. If the antigen has been synthesized by us, we guarantee anti-sera a minimum ELISA-titer of 1:15,000 which is generally sufficient for a meaningful signal in ELISA, WB and staining applications. Please contact us if you need any modification in the protocol or package, like use of species other than rabbit or special handling of the antigen etc.

1. Protein Analysis and Peptide Design: $150
   

2. Standard 70-Day Protocol in Rabbits
   Includes:
   ■ Synthesis of 10 mg of ~85% pure peptide (up to 10 aa long)
   ■ 1 mg of Free peptide to customer
   ■ Conjugation of peptide to carrier protein
   ■ 2 Rabbits (New Zealand White)
   ■ 3 Immunizations/Rabbit
   ■ 1 Pre-immune Bleed
   ■ 2 Test bleeds
   ■ 2 Production Bleeds (20-40 ml sera per project)
   ■ ELISA test
   PRICE: $999.00

3. Includes everything in “Standard” package Plus Peptide-Affinity purification of 20 ml sera.
   PRICE: $1450

4. Includes everything in “Standard” package Except Peptide synthesis and its conjugation. Customer provides a Pre-conjugated, emulsified, ready-to-inject antigen.
   PRICE: $850

Additional Rabbits (each) $350
Additional injections/immunizations $100
Termination of host animal AFTER collecting 25 to 50 ml serum/Rabbit $125
Monthly Maintenance Fee per animal $90
Optional: Conjugation of 3-4mg peptide to KLH/BSA/Ovalbumin (via -SH group) $195
Peptide Design: Analysis and design of two peptides $150

*The price does not include shipping and handling charges. Pre-immune bleed, Production bleeds and “free” peptide are shipped together to the customer in 1 package at the end of project. Additional S&H charges will apply to ship individual items (Pre-immune bleed, test bleed or “free” peptide) before the termination of the project.

Immunization Routine (Standard 70 Day-Protocol)
Day 0: Pre-immune bleed from 2 rabbits & Immunization (FCA)
Day 14: First boost SC (IFC)
Day 28: Second boost SC (IFC)
Day 36: First production bleed + ELISA
Day 40: Second production bleed

With Ready-to-Load samples provided by the researcher, we can run a SDS-gel with one of the various crosslinking percentages chosen by the researcher. We can run as many as 15 samples on 1 gel, stain the gel and provide a digital image. If researcher can not provide Ready-to-Load samples, we can prepare samples from cells and tissues for an additional fee.

With Ready-to-Load samples provided by the researcher, we can run a native gel with one of the various crosslinking percentages provided by the researcher. We can run as many as 15 samples on 1 gel, stain the gel and provide a digital image. If researcher can not provide Ready-to-Load samples, we can prepare samples from cells and tissues for an additional fee.

Alamo Labs offers state of the art gene synthesis service with no set up fee. We try our best to achieve synthesis of difficult genes with sequence repeats or GC rich contents. We codon-optimize gene for expression in organism of your choice as well as sub-cloned it into pUC or pBSK vector with no extra charge. We can sub-clone your gene into vector of your choice for a nominal fee. You'll have to provide vector of your choice.

Test 6: Cell Toxicity of the drug(s):
We can evaluate the effect of a given drug/compound on any cell line of customer’s choice, using a cell proliferation/ viability assay to appraise the anti-proliferative/viability potency of a given drug relative to the other drugs in the same library or to those reported in literature.

Cloning & Sub-cloning Service

Constructs for Adenovirus Expression System
It is a very strong and efficient system for the simultaneous expression of two or more specific gene(s) in mammalian cells. We provide this service for the construction of your gene of interest. Toxin-free final product is ready to transfect HEK cells for the production of recombinant adenovirus.

Constructs for Baculovirus Expression System
One of the most widely used gene(s) expression system for production of recombinant proteins in Eukaryotic cells (such as sf9, Sf21). We offer cloning services for the construction of gene of your interest in baculovirus-based expression vector(s). Final product is ready to transfect insect cells for the packaging of recombinant baculovirus.

Construction of Gene-specific SiRNA
This revolutionary technique to block the expression of a specific protein(s) in cells or animals has proven to be a reliable and quick approach. We'll take care of your SiRNA construct from designing of the specific primers to its cloning in an SiRNA expression vector. We also provide SiRNA duplex synthesis service with desired modifications such as biotin.

Sub-cloning
This service is provided for sub-cloning of gene/cDNA into vector of your choice. For accuracy, recombinant clones will be verified by restriction analysis. A project report with detail analysis and 10 ug of final product will be supplied. Customer supplies the specific gene/ cDNA and vector.

PCR Cloning
We'll design and make gene-specific primers for PCR amplification from a cDNA library. We'll buy commercially available specific cDNA library if customer can not provide it. PCR amplified cDNA fragment will be sub-cloned in TA cloning vector or in a vector of your choice. Recombinant clones will be verified by restriction fragment analysis. For accuracy, two selected clones will be sequenced. A project report with detail analysis and 10 ug of final product will be supplied.

RT-PCR
We'll reverse transcribe and PCR amplify specific gene from customer’s provided total RNA/mRNAs. Amplified fragment will be ligated in TA vector or in vector of your choice. Two clones will be sequenced for the validity of clones. A project report with detail analysis and 10 ug of final product will be supplied.

cDNA Library Construction
We offer cDNA library construction service at very competitive price.

We offer cDNA library construction service at very competitive price. For details please contact us.