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MILKSHAKE: novel validation method for antibodies to post-translationally modified targets by surrogate Western blot.

ABSTRACT: Antibody (Ab) validation is the procedure in which an Ab is thoroughly assayed for sensitivity and specificity in a given application. Validation of Abs against post-translationally modified (PTM) targets is particularly challenging because it requires specifically prepared antigen. Here we describe a novel validation method using surrogate proteins in a Western blot. The surrogate protein, which we termed 'MILKSHAKE,' is a modified maltose binding protein enzymatically conjugated to a peptide from the chosen target that is either modified or nonmodified at the residue of interest. The certainty of the residue's modification status can be used to confirm Ab specificity. This method also allows for Ab validation even in the absence or limited availability of treated cell lysates.

SUBMITTER: Jones KS

PROVIDER: S-EPMC10568613 | biostudies-literature | 2022 Jan

REPOSITORIES: biostudies-literature

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MILKSHAKE: novel validation method for antibodies to post-translationally modified targets by surrogate Western blot.

Jones Kezzia S KS Chapman Amanda E AE Driscoll Holland A HA Fuller Emily P EP Kelly Meghan M Li Xiaofeng X Mansour Sourour S McBride Shannon L SL Zhao Qi Q Weiner Michael M Ferguson Mary R MR

BioTechniques 20211129 1

Antibody (Ab) validation is the procedure in which an Ab is thoroughly assayed for sensitivity and specificity in a given application. Validation of Abs against post-translationally modified (PTM) targets is particularly challenging because it requires specifically prepared antigen. Here we describe a novel validation method using surrogate proteins in a Western blot. The surrogate protein, which we termed 'MILKSHAKE,' is a modified maltose binding protein enzymatically conjugated to a peptide f ...[more]

PMID: 34841898

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