Michelle’s Method of the Month | MILKSHAKE: novel validation method for antibodies to post-translationally modified targets by surrogate Western blot

For January’s Method of the Month I have selected the method presented by Kezzia S. Jones and colleagues in their recent reports article. The method utilizes binding to a surrogate protein (named MILKSHAKE) developed by the authors that combines the target protein peptide with a modMBP (modified maltose binding protein). The authors describe strawberry MILKSHAKE, a version of the method that tests for more stringent antibody validation by including cell lysate when loading the western blot.  Importantly, this method allows for validation of phospho-specific binding endogenous treated lysates, in lieu of phospho-mimicking mutations or less-specific chemical or UV treatments.   

This method is novel, broadly-accessible, and generalizable and provides a method for antibody validation that supports more reproducible research not only for western blots, but also for antibody use in flow cytometry, immunohistochemistry, and immunocytochemistry. 

Method Summary: We have developed a novel method for validating antibodies in a Western blot using modified maltose-binding protein enzymatically conjugated to a target peptide mixed with mammalian cell lysate as the antigen. This method can be used to confirm the primary antibody’s specificity for the target sequence and as a Western blot process control specific to the primary antibody used. We will demonstrate the utility of MILKSHAKE for post-translationally modified (PTM) targets and for determining specificity at an amino acid polymorphic site, but it should be apparent that it can also be used for non-PTMs.  

Read the full article here >>>>