PrecisionAb Validated Western Blotting Antibodies

Antibodies are one of the most important everyday reagents for biomedical science. In recent years, a number of studies have questioned the reliability of published data and highlighted how low-quality antibodies have contributed to a lack of consistent results (Bordeaux et al. 2010; Prinz et al. 2011; Baker and Dolgin 2017).  

We believe antibodies need to be properly validated before they can be trusted for use in scientific research.

What Is Antibody Validation?

“Antibody validation is the experimental proof and documentation that a specific antibody is suitable for an intended application or purpose” (Weller 2018). To address this, the International Working Group on Antibody Validation (IWGAV) has suggested broad guidelines for antibody validation (Uhlen et al. 2016). The IWGAV defined five validation pillars that provide a framework for validation testing. These include genetic strategies, orthogonal strategies, independent antibody strategies, expression of tagged proteins, and immunocapture followed by mass spectrometry (MS).

Bio-Rad’s PrecisionAb Antibody Range

At Bio-Rad, we are committed to providing our customers with reliable research tools, and are constantly raising our standards for antibody quality. The PrecisionAb Antibody range, rigorously tested in western blotting, for specificity and reproducibility, is designed to help researchers produce the highest-quality western blotting data. 

Stringent Validation Process 

PrecisionAb Antibodies are tested against multiple biologically relevant cell lysates that express the endogenous target (Figure 1). Where relevant, negative controls and other important samples such as human serum and human, or rodent tissue lysates are included. For each antibody, we carefully review the literature to identify lysates expected to express the target, and use these to evaluate the antibody. Western blots are carried out by scientists who scrutinize the data comparing relevant bioinformatic database references against signal-to-background ratio, ensuring that only the best antibodies become PrecisionAb Antibodies.  


Fig. 1. A, western blot analysis of whole cell lysates probed with PCNA Antibody (VMA00018) followed by detection with HRP conjugated Goat Anti-Mouse IgG (1/10,000, STAR207P) and visualized on the ChemiDoc MP with 5 sec exposure. Arrow points to PCNA (molecular weight 29 kDa). B, HeLa cells stained with 5 µg/ml Mouse Anti-GPI Antibody (VMA00348, green). Hoechst was used as nuclear stain (blue) and imaged with a 20x objective.​

Using Bio-Rad’s stain-free imaging technology, we monitor protein loading consistency and transfer efficiency.

Transparent Validation Methods

We understand the importance of transparency in our production and testing processes, guiding customers to the correct reagents for their research needs. For this reason every antibody comes with complete antibody information including details of the immunogen used in its generation and recommended conditions for suggested applications, like western blotting. Our western blots are not cropped to narrow the molecular weight range, so any additional bands and off-target effects are never excluded. Reviews of antibodies and information about publications where the antibody has been used are also provided.

For phosphorylation-specific antibodies, we validate by comparing antibody binding in both treated (for kinase activation or phosphatase inhibition) versus untreated cell lysates and by dephosphorylating proteins on western blot membranes (Figure 2). To showcase the different binding characteristics of phospho-specific and total protein antibodies, Phospho-Specific PrecisionAb Antibodies are compared against total PrecisionAb Antibodies. This enables researchers to assess the impact of different treatments and provides a control recommendation.

Fig. 2. Western blot analysis of whole cell lysates probed with A, Mouse Anti-EGF Receptor Antibody (VMA00061) or B, Mouse Anti-EGF Receptor (pTyr1173) Antibody (VMA00752). It is followed by detection with HRP conjugated Goat Anti-Mouse IgG (1/10,000, STAR207P). Membranes were treated with (+) and without (-) lambda protein phosphatase as indicated and visualized on the ChemiDoc MP Imaging System.


Knock-Out Validation

At Bio-Rad, we are taking the necessary steps to enable researchers to perform reliable and reproducible western blotting experiments using our antibodies.

To comply with the IWGAV recommendations, we are implementing additional validation methods recommended by the five pillar validation approach, to further confirm the quality of our antibodies. This includes genetic strategies during which expression of the target protein is compared between wild type and gene knockout using CRISPR/CAS9 (Figure 3 and Figure 4). The datasheet and validation data provided online for each antibody are batch specific.

Fig. 3. Western blot analysis of vimentin CRISPR knockout HeLa (Vimentin KO) and wild type HeLa (WT) whole cell lysates probed with A, Human Anti-Vimentin Antibody (VMA00008) and B, hFAB Rhodamine Anti-Tubulin Primary Antibody (12004166).


Fig. 4. Normalized signal intensity of vimentin from CRISPR knockout and wild type lysates. Signal intensity was normalized to total protein.

Fig. 4. Normalized signal intensity of vimentin from CRISPR knockout and wild type lysates. Signal intensity was normalized to total protein.


References:

  • Baker M and Dolgin E (2017). Cancer reproducibility project releases first results. Nature  541(7637), 269-270.
  • Bordeaux J et al. (2010). Antibody validation. Biotechniques 48(3), 197-209.
  • Prinz F et al. (2011). Believe it or not: how much can we rely on published data on potential drug targets? Nat Rev Drug Discov 10(9), 712.
  • Uhlen M et al. (2016). A proposal for validation of antibodies. Nature Methods 13, 823-827.
  • Weller MG (2018). Ten basic rules of antibody validation. Anal Chem Insights 13:1177390118757462.