How to Perform Protein Quantification for Western Blot Analysis of White Adipose Tissue?
First, the white adipose tissue should be homogenized and lysed to extract total proteins. RIPA buffer is a commonly used lysis reagent, as it effectively disrupts cellular membranes and releases intracellular proteins. Protease and phosphatase inhibitors must be added to the lysis buffer to prevent protein degradation during extraction.
Following protein extraction, quantification can be performed using a BCA protein assay kit. An appropriate volume of protein sample and standard is mixed with the BCA working reagent according to the manufacturer’s instructions. The mixture is then incubated at 37°C for 30 minutes, after which the absorbance is measured at 562 nm using a spectrophotometer. Protein concentrations are determined by comparing the absorbance values of the samples with those of the standards.
Based on the determined protein concentrations, samples are diluted with loading buffer and heat-denatured by boiling. Subsequent steps include SDS-PAGE electrophoresis, transfer of proteins to a membrane, antibody incubation, and signal detection.
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