Protein Analysis

Proteins are large molecules which becomes a difficulty when sepearating!

Size exclusion chromatography is used. 

Large molecule that cannot enter the pores of the chromatography beads elute off the column first. 

SDS PAGE is an electrophoresis technqiue used. 

As prroteins are large molecules, the larger molecules find it difficult to move through the acrylamide mesh and so take longer to move through the gel. 

However smaller particles move a lot quicker. 

Reducing agents can be added to large molecules to break the S-S bonds in the polypeptide.

The protein which contains disulphide bonds is cysteine. 

Standard proteins of molecular weight are subjected to electrophoresis. These marker proteins can be used to estimare the molecular weight of an unknown protein. 

A way to do this is by plotting a graph of logMR versus reltive migration during electrophoresis. This allows molecular weight of the unknown proteins to be read off the graph. 

should be a linear relationship. 

This technique is used after SDS PAGE separation and uses antibodies for the protein of interest. 

After the SDS PAGE, the proteins are transferred to a membrane. 

The membrane is then probed for specific proteins with antibodies. 

It is a scienifc method which determines the arrangement og atoms of a crystalline solid in three dimension space. 

crystal --> diffraction pattern --> electron density map --> protein model