Validating commercial Validating commercial antibodies in the most antibodies in the most

cost-effective waycost-effective way

AIF1 / ABI1 antibodies

Catalogue items 12 March 2014

Examples of Antibodies on the market appearing as different but they are not

protein Biocompare (suppliers) actual # different AbAIF1 / ABI1 348 (31) ~30PIC3CA 220 (35) ~32FOXP3 1055 (47) ~31NANOG 574 (42) ~45OCT4. 582 (41) ~30

PIK3CA # frequencyAcris 16AOL 10LSBIO 10Abcam 9 7./4Abnova 6 5./1Novus 6 4./3./2Thermo 6Abgent 5 9./5Sigma 5Biorbyt 5GeneTex 4 7./6ProteinTech 4 4./2Aviva 3CST 3 2./1./1Mybiosource 3Millipore 2 1Origene 2 1./1SantaCruz 2 1./1EverestBiotech 1 16ABDSerotec 1ProSci 1

AIF1/ABI1 ACRIS 24 products

species antigen manufr frequencygoat aa81-93 1 5goat aa70-80 1 4goat aa28-42 1 5

rabbit protein 2 1rabbit aa13-44 2 1rabbit aa85-147 3 1rabbit peptide 3 1rabbit peptide 4 1mouse mab#10225 2mouse mab#2A2-B6 5 1

• Other External Manufacturer

Welcome to the world of OEM

• One manufacturer, one product with many brands• QC data responsibility of rebranding vendor• Batch-to-batch variations ignored by many vendors• QC data presented from previous animals• QC data presented from own customers• QC data presented from own QC department• Rebranding vendors buying from each other• Same product appearing several times on same catalogue

Polyclonal Antibodies

Entire protein as antigen

Specific region as antigen

Peptide as antigen

Specific peptide antibodies

Undisclosed peptide as antigen

Antigen affinity purified

C-terminal peptide as antigen

IgG purified

Disclosed peptide as antigen

Antigen affinity purified EB12421 + EB12422

Novus Biologicals NB120-3446

Rabbit anti-NFAT5 (DLLVSLQNQGNNLTGSF)

Antibodies on line ABIN747683

Rabbit anti-NRSF / REST

Calc MW 120kDa

Everest Biotech

CAPG antibodies

on U937, spleen and PBL

WB problems

DDAH1 antibody EB05278

HTR5A antibody EB09577 Non-specific signals in human hippocampus, substantia nigra and

temporal cortex

~40kDa expected

Secondary-derived 55kDa background

10 min exposure 1 min exposure

Discrepancy between assays

CCK2R antibody EB06767

Calc MW 48.4kDa

EB06767 (4µg/ml) staining of paraffin embedded Human Pancreas. Steamed antigen retrieval with Tris/EDTA buffer

pH 9, HRP-staining.

Figure 1 in El-Kouhen K, Morisset J., Int J Endocrinol. 2009;2009:875167.

PMID: 20011057

Statistics in Everest database

IHC with or without WBtotal IHC IHC IHC+WB

641 278 363% 43.4 56.6

Independent quality checks

Everest Biotech

Abcam

Antibodies on line

Antibody Resource

Free antibody in return of validating data for the product sheet

Discounts in return of validating data for the product sheet

Independent quality report (through Science Exchange program)

Assessments in labs at Cambridge and Oxford Universities

OneWorldLab Buy 1/10th size sample for $50, submit your conclusions

pAbmAbs Buy and try from any vendor and submit your conclusions

Scientific citationsReferences on the vendor’s datasheets

Antibodypedia, Biobrea, CiteAb

Warning!! Citations are retrospective, ignoring batch-to-batch variations

• For low usage: buy from large vendor with many choices from different manufacturers

How to shop around for antibodies?

• For high usage and assay developers: buy direct from the manufacturer• Watch out for defined antigen (sequence of immunizing peptide), or defined

region of protein

• Watch out for clone numbers• However, clone numbers may have changed by the vendor• Verify data consistency between literature and what is claimed by the

vendor

Antibodies

Monoclonal Antibodies

• Relate price to the quantity of the offered antibody

• Validate in the required assay, not in another one.