Cleaning Up Western Blot Signals From Immunoprecipitated Samples Using Alternative Detection Methods

Abstract

High background signal is a common problem experienced when detecting proteins isolated through immunoprecipitation (IP) by Western blotting (WB). The most frequent cause of high background is signal interference from the heavy and light chain fragments of the denatured immunoprecipitating (or capture) antibody ' by-products of IP labeled by species-specific secondary antibodies at the WB stage. Here we comment on alternative methods for the detection of immunoprecipitated proteins by WB that avoid labeling of the heavy and light chain to varying extents. Certain methods have been described elsewhere (1), however, their use remains less widespread than traditional detection methods despite offering the researcher considerable advantages.

Characterization of a novel modification of a CHO-produced mAb: Evidence for the presence of tyrosine sulfation

“…It has been shown in the past that denatured heavy and light chains can bind detection antibodies non-specifically. 20 As a control for cross reactivity with phosphorylation, lane 1 was loaded with a commercial source of EGF-treated A431 cell extract that is enriched with phosphorylated proteins. The lack of positive signal in lane 1 of the lower panel shows the anti-sulfotyrosine mAb does not have strong cross reactivity with phosphorylation ( Fig.…”