Protein concentration in the analyzed sample should be estimated before the sample is loaded on a gel

Surprisingly, many laboratories do not have the crucial step of analyzing the protein concentration included in their protocol. Protein is often extracted from a range of samples, and loaded on the gel based on volume, rather than protein concentration. This would work if each sample was extracted with exactly the same efficiency, but this is not the case; especially when protein extraction is done manually. Accurate protein quantification is crucial in securing reproducible and reliable data when doing Western blot analyses. 

Some assays used to measure protein concentration are:
  • Biuret method
  • Bradford
  • Lowry
  • BCA assa
  • UV absorbance at 280 nm

The following parameters should be considered before choosing a specific assay:
  • Compatibility between the sample and the components of the applied extraction buffer
  • Assay range
  • Speed and ease of conducting the assay
  • Requirement of a spectrophotometer or an ELISA plate reader. 

    What to load in a well?

There is a wide range of assays, available from different suppliers, which are compatible with components commonly used in extraction buffers, like detergents, urea, TRIS and HEPES. In case of photosynthetic tissues, loading can be done based on chlorophyll content, which can be easily estimated following the extraction. 

Loading/protein or chlorophyll concentration is highly recommended!