Product Validation

As a company of scientists, we understand how important it is to work with products that have been validated for specificity, selectivity, activity, stability, and performance.

We work hard to ensure that every product is validated in multiple research applications, and with samples derived from multiple species, and we share this data on our website. Our validation process uses a variety of techniques, including: western blot, immunocytochemistry, ELISA, flow cytometry, immunofluorescence, protein arrays, immunoprecipitation, and chromatin immunoprecipitation.

The reactivity of an antibody with homologues from different species can be a desired feature, making the product more versatile, or an unwanted side-effect. We share all of our specificity data on our website enabling you to make informed purchasing decisions.

Figure 1: Anti-AIF1 Antibody (A82670) (1µg/ml) staining of Human Frontal Cortex (A), Rat Brain (B), Mouse (C) Brain and Pig Brain (D) lysates (35µg protein in RIPA buffer). Detected by chemiluminescence.

Figure 2: Western blot analysis of whole brain tissue lysates using Anti-MAP2 Antibody (A85363) (1:50,000, green): [1] protein standard (red), [2] adult rat brain, [3] embryonic E20 rat brain, [4] adult mouse brain.

An increasing number of our antibodies are now tested for specificity and selectivity against 19,000+ full-length human proteins on human genome-wide protein arrays. These arrays compare the binding of an antibody to a target protein and the binding to off-target proteins providing a complete overview of the specificity and selectivity of an antibody. With our protein array validated antibodies, researchers can be confident they're using precision tools.

Figure 3: Analysis of a protein array containing more than 19,000 full-length human proteins using Anti-beta III Spectrin Antibody [SPTBN2/1584] (A253226). Z-Score and S- Score: The Z-score represents the strength of a signal that a monoclonal antibody (MAb) (in combination with a fluorescently-tagged anti-IgG secondary antibody) produces when binding to a particular protein on the HuProt™ array. Z-scores are described in units of standard deviations (SD's) above the mean value of all signals generated on that array. If the results for the targets on the HuProt™ array are arranged in descending order of the Z-score, the S-score is the difference (also in units of SD's) between the Z-scores. S-score therefore represents the relative target specificity of a MAb to its intended target; a MAb is considered to be specific to its intended target if the MAb has an S-score of at least 2.5. For example, if a MAb binds to protein X with a Z-score of 43 and to protein Y with a Z-score of 14, then the S-score for the binding of that MAb to protein X is equal to 29.

We have incorporated knockout validation, using CRISPR/Cas9, as part of our standard antibody validation process. The knockout validated seal indicates that an antibody has had its specificity confirmed by knockout validation.

Figure 4: Western blot analysis of extracts from normal (control) and ELAVL1 knockout (KO) 293T cells, using Anti-ELAVL1 Antibody (A13545) at 1:1,000 dilution. Secondary antibody: Goat Anti-Rabbit IgG (H+L) (HRP) (AS014) at 1:10,000 dilution.

Figure 5: Western blot analysis of extracts from normal (control) and S100A4 knockout (KO) HeLa cells, using Anti-S100A4 Antibody (A13562) at 1:1,000 dilution. Secondary antibody: Goat Anti-Rabbit IgG (H+L) (HRP) (AS014) at 1:10,000 dilution.

We encourage all our customers to submit product reviews and share what works and what doesn’t. All reviews are published online, allowing scientists to learn about product performance, new applications, optimal dilutions, and to see images of our products at work.

Figure 6: Immunofluorescent analysis of primary cultured astrocytes using Anti-GFAP Antibody (A85419) (green) and DAPI nuclear DNA staining (blue). Data submitted in a customer product review.

Figure 7: Western blot analysis of extracts from rat brain tissues probed with Anti-mTOR Antibody (A27028) (Lane A), Anti-FoxO3A Antibody (A26998) (Lane B), Anti-AKT (phospho S473) Antibody (A27292) (Lane C), and Anti-NLRP3 Antibody (A14882) (Lane D). Data submitted in a customer product review.