In our PrecisionAb range of western blot antibodies, we provide mono- and polyclonal antibodies that have been rigorously tested on multiple cell and tissue lysates. The results are available on the corresponding data sheet for each antibody. Only antibodies that perform according to our strict performance criteria (see below) get the PrecisionAb label. We are now in the process of further validating PrecisionAb Antibodies to scientifically prove target binding in western blot, as outlined here.
Our methodologies include:
In accordance with recommendations by the Internal Working Group for Antibody Validation (Uhlen et al. 2016), we use various methods to validate PrecisionAb Antibodies.
These include genetic strategies such as measuring the relevant signal in control cell lines, where the target gene has been knocked out by CRISPR-Cas9 (Figure 1).
Fig. 1. Western blot analysis of cell lysates from target-knockout and wildtype cells. eHAP whole cell lysates probed with anti-LPP (lipoma-preferred partner) antibody followed by detection with HRP conjugated Goat Anti-Mouse IgG (1/10,000, STAR207P) and hFAB Rhodamine Anti-GAPDH IgG (1/1,000, 12004167) and visualized on the ChemiDoc MP Imaging System with 40 sec (LPP Antibody VMA00287) and 10 sec (hFAB Rhodamine Anti-GAPDH IgG, 12004167) exposures. The arrow points to LPP (molecular weight 75 kDa) and to GAPDH (molecular weight 37 kDa).
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).
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), followed by detection with HRP conjugated Goat Anti-Mouse IgG (1/10,000, STAR207P). A431 cells were treated with or without PVD (sodium pervanadate), and membranes were treated with (+) and without (-) lambda protein phosphatase as indicated and visualized on the ChemiDoc MP Imaging System. Arrows indicate the MW of the target (175 kDa).
To assess antibody performance we analyze multiple cell lines and tissues which are known to express the target, as well as negative controls. We publish the results as images on our website and on the antibody datasheets.
We recommend dilutions, reagent optimum operating conditions, and protocols to guide you with your experimental design and save you time. We show the full blot image from our in-house testing and therefore enable researchers to assess the antibody performance for themselves. We also include reviews of antibodies and information about publications where our antibody has been used.
All PrecisionAb Antibodies have their own specific conditions for optimum performance. These include concentration, choice of secondary antibody, dilution and storage conditions as well as information on the target lysate and expected outcome of quality control testing. By testing each PrecisionAb Antibody against its own optimized criteria for western blotting, we can ensure that they perform consistently under those conditions and generate reproducible results.