Protein Overexpression
 
The validity of a target protein’s endogenous signal can be further substantiated through overexpression of that protein using a recombinant construct. This approach offers supportive evidence verifying antibody performance and gives greater confidence that the detected signal is not artificial. In addition, the overexpression of related proteins allows evaluation of an antibody’s possible cross-reactivity with homologous targets. The following examples show how we use protein overexpression to validate antibody quality.

 

 
 

 

 

To analyze the specificity of our NF-κB p65 antibody (GTX107678), a western blot was performed on both HeLa wildtype (WT) and CRISPR-generated RELA knockout (KO) lysates. As expected, the NF-κB p65 signal was not present in the KO lysate. Alternatively, lysates from 293T cells that overexpress GFP-tagged NF-κB p65 were examined, and the signal was detected at its predicted molecular weight. The combined data provide both negative and positive controls to validate the same antibody.
 

NFkB p65 antibody

 

LC3B antibody

 

 

 
 

 

In another example, to query potential cross-reactivity of our MYH10 antibody (GTX634160) with homologous proteins, lysates from 293T cells expressing either EGFP-tagged MYH9, MYH10, or MYH14 were subjected to western blot. As shown below, the MYH10 antibody (GTX634160) only detected overexpressed MYH10 and not MYH9 and MYH14. Similarly, our IDH2 antibody (GTX133078) only detects IDH2 protein but not IDH1, highlighting the utility of this antibody for studying IDH2-specific biological events.
 

MYH10 antibody [GT246]

 

IDH2 antibody

 

 

GeneTex antibodies that have been validated using the “Protein Overexpression” strategy will be labeled with the icon in relevant product pages and marketing materials.