How does trypsin help investigate protein location and structure in a SDS-PAGE? Or rather how would it affect the results of a SDS-PAGE as opposed to a SDS-PAGE without trypsin treatment? Thank you.

Wow, who would have ever thought I would answer this one here. My PhD happened to be on the Signal Recognition Particle Receptor and we used this assay regularly!

Basically, you target the protein to vesicles and then treat them with trypsin (or not -- the control) then run them on SDS-PAGE. If the proteins are inside the membrane vesicles then they are protected from the trypsin and you see them on the SDS-PAG, at the same size as the control. If they are not protected (ie not translocated into the vesicle) then they are degraded by the trypsin and you don't see them on the gel.

There are many variations on this, and it can be done either using vesicles (as I did, with in vitro transcription/translation systems) or using live cells and fractionation. Furthermore, you can label certain parts of the protein and, with transmembrane proteins, use trypsin protection patterns to figure out the topology of the protein in the membrane.


And... as far as protein structure goes, you can use a more limited trypsin proteolysis to identify tightly folded domains vs. regions of the protein that are more loosely folded. That is, the trypsin will target the more loosely folded regions first, digesting the protein into separate domains, which can then be determined by SDS-PAGE (and often immunoblotting with antibodies to certain epitopes of the protein).