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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