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

Cell lysis is an essential step in the extraction and purification of intracellular proteins, and the method that you use to disrupt your cell samples can determine the success and reproducibility of your downstream applications to a great extent. Keep in mind that one vital component of the starting biological material is inevitably lost in every step of the experiment, so it’s important to get everything you need right from the start. This underscores the need of choosing the cell disruption method that will allow you to extract the best possible yield and purity of your biological materials.

Protein extraction from plant, animal, fungi, bacteria, etc is well known using different techniques. Many buffers are used to solubilize water-soluble proteins. There are few proteins which are insoluble and are rigid to be extracted by physical means or they lose activity when extracted by denaturants. One of the promising methods is to use organic solvents to extract proteins inactive state. C4 and C5 alcohols (butanol, pentanol) are mostly used for the purpose as they act as differentiating solvents. When an organic solvent is added to a protein solution followed by ammonium sulfate, the solution separates into three phases. Upper organic phase and lower aqueous phase, depending on the concentration of ammonium sulfate some proteins precipitate out and form an intermediate layer.

Immunocytochemistry is one of the most commonly used methods for protein localization in the cells or tissues using specific antibodies. The overall credibility of the results from Immunocytochemistry depends on two factors:

Mass spectrometric analysis is one of the important technique in protein biochemistry and peptide analysis. It is used to identify and analyze proteins and peptide samples. Care should be taken for sample preparation for mass spec analysis as the quality of data depends on the purity of sample applied. A variety of strategies are used to prepare the samples for mass spectrometric analysis.