EGFR, Santa Cruz Biotechnology, sc-03

We generated a top 1000 list of the most studied and most published proteins, and analyzed the literature validation data for the commercially available research antibody tools. EGFR is the third most studied gene on the list. We identified three publications providing strong validation for the use of sc-03 in WB and IF. Mannsperger et al., 2010, Proteome Science entitled RNAi-based validation of antibodies for reverse phase protein arrays. Paper compares two anti-EGFR antibodies (antibody 1 (sc-03) and antibody 2 (Cell Signaling 2646)) in Western blotting following EGFR siRNA knockdown in MDA-MB-231 human breast cancer cells. Negative control is non-targeting siRNA. ~80% reduction after EGFR silencing; predominant ~175 kDa band plus weak ~60 kDa band. Signal lower than antibody 2 (Figs. 1,2). Specific enough to report the expected knockdown by WB, although a minor additional band was present. The Santa-Cruz sc-03 antibody recognizes a C-terminal epitope. The sc-03 antibody was validated in several applications in Brand et al., 2014, Molecular Cancer Therapeutics entitled Nuclear epidermal growth factor receptor is a functional molecular target in triple-negative breast cancer. The sc-03 antibody showed specific staining of triple-negative breast cancer cell lines (TNBC) as visualized using confocal immunofluorescence and validated by EGFR siRNA knockdown + blocking peptide competition + secondary-only control (Fig. 1B) EGFR signal was strongly reduced by EGFR siRNA and by preincubation with blocking peptide; no signal with secondary-only control. This provides strong validation for IF detection/localization of EGFR, including nuclear signal in the tested TNBC models. The IF observations were confirmed by Immunogold labeling of nEGFR in EM, and by IHC staining for EGFR on a total of 74 TNBC patient tumor sections. However, no independent controls were provided for EM and IHC. Scheving et al., 2015, American Journal of Physiology - Gastrointestinal and Liver Physiology entitled Loss of hepatocyte EGFR has no effect alone but exacerbates carbon tetrachloride-induced liver injury and impairs regeneration in hepatocyte Met-deficient mice. The authors leveraged a hepatocyte-specific EGFR knockout mouse model to validate the sc-03 antibody. Liver sections from HS-EGFRKO and wild-type mice were analyzed. Hepatocyte EGFR staining was essentially lost in knockout liver; isolated hepatocytes and nonparenchymal/bile duct cells retained staining. Authors explicitly describe KO sections as useful for antibody specificity validation. Fig. 2A; Immunohistochemical localization of EGFR Very strong IHC specificity evidence because knockout hepatocytes are surrounded by genetically unaffected internal positive cell types. One of the clearest primary-literature examples of KO-based EGFR IHC validation. The authors further validated sc-03 in Western blotting using mouse liver lysates from wild-type and HS-EGFRKO mice as samples. Equal loading verified by Ponceau S. Immunoblots showed strong loss of hepatocyte EGFR expression in the knockout model and were used alongside IHC to establish deletion. Western blotting methods / Results around Fig. 1-2 Direct genetically defined WB evidence for sc-03 in mouse liver. We rated sc-03 based on the available information and according to the pAbmAbs criteria.