Affinity Chromatography
Blogging is so fun, I can write all kinds of pointless stuff that I enjoy and no one else cares to ever read. So I’ll take some time to share my knowledge of biochemistry, since I know the world is eagerly awaiting my contribution to the internet:
Affinity chromatography is used to purify mixtures of unknown proteins for a single protein of interest. The column matrix contains adenine monophosphate(AMP) immobilized on beaded agarose. The protein sample is diluted before running through the column to bring it down to a measurable range ( < 2 A280). When the protein sample is run through the column, lactate dehydrogenase(LDH) binds to the AMP and becomes trapped in the column. The protein sample is followed by elution buffer to “wash the column” and elute out the contaminant proteins that have no affinity for AMP. Next a low concentration solution of nicotinamide adenine dinucelotide(NADH) is run through the column to elute out the proteins that do not bind strongly to the AMP. This “low NADH” is followed by a “high NADH” solution whose NADH concentration is high enough to elute out the LDH. Fraction collector tubes are used to collect a specified volume of solution as it elutes from the column. Measuring absorbance values of these collector tubes at A280 allows us to detect the presence of aromatic amino acids, such as phenylalanine, tryptophan, and tyrosine and therefore estimate the amount of protein eluting from the column, following the “washes”.
