The Protein Man's Blog | A Discussion of Protein Research

Proteomes can be made up of more 15,000-20,000 different protein species that differ greatly in properties such as molecular mass, charge, abundance, hydrophobicity and other characteristics. Extraction of proteome for analysis is therefore a daunting task that requires careful attention to details. Sample preparation for proteome analysis requires solubilization of proteins. The choice of optimal method for solubilization of protein mixture is critical since sample preparation affects the outcome of analysis. Usually the steps involved are homogenization, solubilization and stabilization when proteome sample is prepared from cells or tissues.

The RIPA buffer, or Radioimmunoprecipitation assay buffer, is ideal for whole-cell extracts, as well as protein assays. However, it is an essential component in other applications too. 

Successful biochemical analysis heavily relies on the effective extraction of biologically active proteins from source materials (e.g., cell and tissue samples). Thus, you need to have an excellent working knowledge of your target protein(s) and use the appropriate extraction buffers for a given experimental design to ensure optimal protein recovery.

When it comes to solubilizing and denaturing proteins prior to isoelectric focusing and 2-D gel electrophoresis, most researchers choose urea. This is not surprising since this mild chaotropic agent can completely disrupt the interactions between protein molecules and increase the efficiency of protease activity in protein digestion while effectively preventing proteins from aggregating and/or precipitating. In addition, urea is also used for renaturing proteins from previously denatured samples.