Proteomics/Protein Sample Preparation/Prepts for Chromatography

Preps for Chromatography

• Solubilization and Solution Preparation

For High Performance LC (HPLC), it is very important that the sample solution (mobile phase) be free of particulate matter. Due to the sensitive nature of a HPLC column and the extreme pressures involved, such contaminants can be extremely damaging and Protein_Sample_Preparation|Contaminant_Elimination#Contaminant_Elimination is very important.

• Recombinant techniques for affinity chromatography

Affinity chromatography is a protein separation technique where substrates with affinities for specific proteins are used in the solid phase of a chromatography column. One highly specific technique is to use recombinant technology to isolate proteins expressed from recombinant DNA. If a gene is inserted into a cloning vector for the purpose of fermenting large amounts of a protein, the gene can be altered to include additional amino acids in the protein. This subsequence can then be utilized as a binding site for the stationary phase substrate. Two examples of this are the additions of histidine residues that bind covalently to certain metal ions and the addition of glutathione-S-transferase protein that will bind with glutathione. http://en.wikipedia.org/wiki/Affinity_chromatography

Single-step purification of bacterially expressed polypeptides containing an oligo-histidine domain. Gene. 1992 Feb 1;111(1):99-104.

Solubilization and purification of enzymatically active glutathione S-transferase (pGEX) fusion proteins. Anal Biochem. 1993 Apr;210(1):179-87.