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

While detergents play a vital role in separating proteins from the hydrophobic portions of the cell membrane during extraction and sample preparation, they need to be removed after successfully serving their purpose to prevent them from interfering with downstream applications (e.g., IEF, ELISA, protease digestion of proteins).

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.

Protein cross-linking reagents, fondly called “cross-linkers” by researchers, are molecules with two or more reactive ends that can attach themselves to specific functional groups (e.g., primary amines, carboxyls, sulfhydryls, carbohydrates, and carboxylic acids) on proteins and other biomolecules via a covalent bond.