MILKSHAKE: novel validation method for antibodies to post-translationally modified targets by surrogate Western blot.
Biotechniques
; 72(1): 11-20, 2022 01.
Article
en En
| MEDLINE
| ID: mdl-34841898
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.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Proteínas
/
Anticuerpos
Tipo de estudio:
Diagnostic_studies
Idioma:
En
Revista:
Biotechniques
Año:
2022
Tipo del documento:
Article
País de afiliación:
Estados Unidos