Protease: Post Electrophoretic Reactivation - (Mar/28/2006 )

Hi everybody,

I'm working on leaf protease of a plant found in our part of the world. During my literature search I found an article describing the detection of proteolytic activity in a certain bacteria after SDS-PAGE. The title grabbed my attention and I was willing to try it out but after going through the paper, I was staring at a whole lot of questions. I would be happy if you people could help me out since I am just starting as a researcher, this being my undergrad project work.

1. The particular protease was from Pseudomonas and I presume bacteria have plenty of them unlike plant and moreover leaf. So will this method work?

2. SDS-PAGE as I know serves the purpose of coating uniformly the protein with negative charges so that they are separated solely on the basis of their molecular weight. In this paper, they don't heat treat the samples. Why is this? SDS is there in the sample buffer but they didn't heat the samples. Am I right in thinking that this is done so as to retain the activity of the protease since it's still in its native conformation? Then why not to omit SDS in the first place and perform native PAGE and see the proteolytic activity after incubating with the substrate?

3. After completing the run, the gel was washed in 67mM glycine-NaOH buffer pH 9.0 containing 10% methanol and then rinsed in the above buffer without methanol. The paper says it's done for the partial removal of SDS. How come??

4. Now the gel was placed in a solution containing substrate ie 0.5% casein in 67mM glycine-NaOH buffer pH 9.0 for one and half hour. Then the solution was decanted, rinsed in 67mM glycine-NaOH buffer and finally Coomassie blue staining was performed. The proteolytic activity is seen as clear areas against a blue-stained background. Why does glycine-NaOH buffer prop up everywhere? I never encountered this combo? What does it do? Do you seriously think with the little protease in plant leaf, would I be able to detect the clear areas on the gel? Moreover is it possible to try out other substrates such as BSA?

I'm sorry for the length. But please you could read it at your leisure and any suggestion would be helpful. I would be also talking to my supervisor. But your any sort of contribution would be worthwhile.

1. it may work. you won't know for sure unless you try.

2. sds also unfolds the protein so that the gel is (theoretically) separating rods. the sample is not heated because, in some cases, it is not always necessary. when running normal(?) sds-page i only have to heat the sample for some recalcitrant proteins otherwise i normally don't. the protein is not active and not in its normal conformation. you could do this with a native gel but you won't see the same type of separation.

3. the methanol is used to remove sds from the gel (to remove from gel doesn't absolutely require meoh but it helps) and to remove enough sds from the protein to allow for partial refolding (hopefully enough to bring the active site back into operation).

4. that is glycine buffer adjusted to pH 9 with naoh. apparently these are the conditions required for optimal activity of the protease that these people are working with. you could use a different buffer/pH. it should work for relatively small amounts of enzyme. you can use other substrates as long as they bind cbb when whole and not when they are chewed up (casein is actually a pretty good general substrate for proteases).

hope this is useful for you

The name for this type of gel is a zymogram, which may help you in locating other literature and protocol references.