How to Effectively Fractionate Proteins: A Guide to the Best Methods

Nuclear & Cytoplasmic Extraction Kit Scheme

Protein fractionation refers to the process of isolating, identifying, and characterizing various proteins present in a sample. Proteome analysis is often hindered by the presence of high-abundance proteins, which can overshadow the detection of low-abundance proteins. These low-abundance proteins are frequently the most interesting. Effective protein fractionation techniques are essential for isolating these proteins and enabling comprehensive proteomic studies.

Protein fractionation can be based on various properties such as size, shape, solubility, stability, sedimentation rate, binding capacity to ionic groups, and affinity for substrates. Additionally, proteins can be separated based on their cellular localization: cytoplasmic, nuclear, or membrane-bound.

Fractionation by precipitation can be achieved through methods such as “salting out” with ammonium sulfate, isoelectric precipitation, or using solvents like alcohol or acetone. These techniques exploit differences in protein hydrophobicity and isoelectric points to selectively precipitate unwanted proteins, leaving the proteins of interest in solution.

Proteins can also be fractionated using chromatographic and electrophoretic procedures. Different proteins can be separated using gel filtration, which separates proteins based on their size and shape, and/or adsorption chromatography.

Recent advances in protein fractionation include the use of magnetic particles, which offer rapid separation. Magnetic particles can be functionalized with various agents such as ionic metals, polymers, biomolecules, and antibodies to precisely recognize and bind proteins.

There is no single method for protein separation that suits all cases. The choice of protein fractionation method depends on the specific objectives of the research. Combining multiple techniques is often necessary to achieve successful protein isolation and analysis.