Proteomics/Protein Sample Preparation/Prepts for Chromatography

Preps for Chromatography
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.
 * Solubilization and Solution Preparation


 * 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.