Western Blot Methodologies for Analysis of In Vitro Protein Expression Induced by Teratogenic Agents.

Western Blot Methodologies for Analysis of In Vitro Protein Expression Induced by Teratogenic Agents.
Methods Mol Biol. 2018;1797:191-203
Authors: Martins-Gomes C, Silva AM
Abstract
Western blotting permits immunodetection, characterization, and quantification of proteins in cell (or tissue) homogenates. It also enables detection of protein modification (e.g., phosphorylation) or degradation (e.g., hydrolysis), even at low abundance. Sodium dodecyl sulfate (SDS)-polyacrylamide gel is used to separate proteins from homogenate which are then transferred electrophoretically to polyvinylidene difluoride (PVDF) membranes. After membrane "blocking," to reduce nonspecific binding, proteins of interest are detected using specific antibodies (antigen detection), which are then bound to a secondary antibody linked to a label (e.g., fluorescent, chemiluminescent, or chromophore). After signal detection and acquisition, quantification of the resulting bands is achieved using densitometry software. Results are normalized against controls and housekeeping proteins (e.g., GAPDH, beta-actin and tubulin), which are constitutively expressed proteins that maintain cell viability. This chapter outlines the use of the Western blot technique optimized for the in vitro analysis of changes in the protein expression induced by teratogenic exposure.
PMID: 29896693 [PubMed - in process]

Source: Mol Biol Cell - June 15, 2018 Category: Molecular Biology Authors: Martins-Gomes C, Silva AM Tags: Methods Mol Biol Source Type: research