Question

Describe the chromatofocusing process including a) how separation is enacted b) what pH conditions you need and why c) some applications of this technology d) contrast this with IMAC e) what applications might work with IMAC that may not work with ion exchange or chromatofocusing and why? Please list references when answering

Answer 1

a) The technique is used for improperly purified samples. This chromatography technique is used for purification of proteins. The medium used for the separation of the proteins is buffered at suitable pH (above highest pH required) The elutant is also buffer as per the lowest pH required. When the buffer is allowed to flow over the column, the pH gets lowered, generating pH gradient. The proteins start separating, with the start of protein with PI less than the pH of column. The proteins start getting positively charged and being repelled by the column and is separated.

b) The pH conditions maintained according to the isoelectric point of protein

c) The techniques is used for separation of protein

(i) In bacterial analysis

(ii) in enzymatic analysis

(iii) enzyme separation obtained from microbes

d) IMAC :Immobilized-Metal Affinity Chromatography : It works on the principle of metal ion effeciency towards amino acids. The main amino acid used will be histidine. The column of chromatography is packed with metal ions and when amino acid are passed over it, some amino acids show effeciency for the packed metal ions while the amino acids which do not show affinity are separated out

e) In case of chromatofocussing, the pH is used according to the PI of proteins however some amino acids gets precipited near to their isoelectric point so we can use IMAC technique.

You may use the following review article for better understanding

http://molbiol.ru/forums/index.php?act=Attach&type=post&id=181413