Identify the component of multiprotein complex _2

Technique for purification of test protein -
* tandam-affinity purification (TAP)-  Develope as means of studing protein complex in s.cerivisiae           -gene for test protein is modified so that the c-terminal end of  protein contain binding site of another protein called calmodulin.
 -cell extract is prepared under gentile condition so that  multiprotein do not break,
 - now extract is pass through affinity column containing calmodulin. so protein containing c-terminal calmodulin is retain in column and purify.
Technique for purification and identification of intact complex protein -
*multi-dimensional protein inditification technology (mudPIT)- it combine various chromatograpy technique such reversed phase liquid chromatography and cation exchange rasin in order to isolate intact protein complex and component of these are identify by mass chromatography.

Identifying proteins that intract with one another

Most powerful technique is - yeast two hybrid system and phage display.
limitation of these two technique-
1) fail to identifying the component of multiprotein complex or require a long series of two hybrid experiment, because multiprotein is made up of core protein and ancillary protein and these ancillary proteins may be bind at different portion of core protein so these ancillary protein does not interact with each other in yeast two hybrid experiment.
2) large protein are displayed insufficiently in phage display because they disrupt the phage replication cycle.


                                                  So affinity chromatography is used. In this, the test protein is attached to chromatography resin and placed in column. Cell extract is pass through the column in low salt buffer( in low salt buffer H bond is formed which hold proteins together in a complex. the protein that interact with the test proteins are bounded and rest are washed away. The interacting protein are then eluted in high salt buffer.
Disadantage of affinity chromatography -
- need to purify test protein.
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