protease bands on the gel.... - should they be visible? (Sep/29/2006 )
i digest my purified protein with protease. as it is purified and bound to GST beads i run PAGE to check for truncated protein. so i still see my native protein and almost same intensity bands of the protease 
...my professor says i shouldn't be able to see the protease bands as it is the enzyme (am not quiet sure i understand the meaning) and if i am seeing the bands of the protein and that of the protease almost the same it simply means that i added too much of protease. and to add to my misfortune i think that the band of truncated form coonsides with that of the protease band.
please i want to hear from those who did proteolysis; can you see the protease band on PAGE?
...my professor says i shouldn't be able to see the protease bands as it is the enzyme (am not quiet sure i understand the meaning) and if i am seeing the bands of the protein and that of the protease almost the same it simply means that i added too much of protease. and to add to my misfortune i think that the band of truncated form coonsides with that of the protease band. please i want to hear from those who did proteolysis; can you see the protease band on PAGE?
If you are sure that you have washed the GST beads real well, then there is a good possibility that the digested protein has the same MW as the enzyme. Have you confirm this with WB, or run a native gel see if you get a single band?
thank you gene hunter, im running WB now so let's see how it goes. i washed the beads 3 times with PBS dont know of that is enough though... 
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please can you explain more.....why should I see only one band on native PAGE?
may be for the native gel, your active protease work on the protein and digest it ,so only single band is supposed to appear on gel
is there also a control running without GST proteins but with dummy protein and without, for each utilizing proteolysis protocol?
if one knows the protease (if it is not part a kit without good information) one knows the molecular weight; so one knows where its protein of interest should run;
visibility of polypeptides in gel depends not only on conc of proteins but also on staining method; e.g. low nanograms will not be stained by CBB
if truncated protein and protease really have the same Mw you may separate them by 2D GE;
I agree that native PAGE is more protective against proteolysis as SDS PAGE; SDS sample buffer denaturates proteins which are better catalysted by proteases than native proteins
what your professor probably means is that you only need to add a very small amount of the protease to digest your protein. if you add just what you need for the reaction conditions and time then it will probably be undetectable with coomassie staining.
the thing that worries me is that native band is not getting any smaller...
i think that would mean that the protease is not digesting very well, so decreasing the protease amount will only make things worse no?also i have only one vial of protease left and they say dont thow refreeze. so it is my last chance, but frankly i don;t know what to do
What about reactions conditions/temp?
i think that would mean that the protease is not digesting very well, so decreasing the protease amount will only make things worse no?also i have only one vial of protease left and they say dont thow refreeze. so it is my last chance, but frankly i don;t know what to do
if you are not seeing significant proteolysis then you may have to switch to a different protease or adjust your reaction conditions (and/or the condition of your protein).
i am using same conditions and same protease used before by my freind. however she had cell lysate and i have purified protein bound to GST beads. i dont even know the concentration. you can't actually know how much is bound to the beads