SDS-PAGE and proteolysis - (Sep/05/2008 )
Hello to everybody!
I recently started doing some proteomics and right now I have some troubles in running preparative SDS gels. Indeed, I often find proteolytic products spread through the whole gel.
The protocol I am using now is the following:
I break yeast cells by bead-beating in a lysis buffer similar to the sample buffer containing:
0.1M Tris pH 7.5
10% glycerol
Protease inhibitors (Roche)
1mM PMSF
2.5% SDS
Before loading the extract on a gel I just add sample buffer and heat the mix at 95°C for 5’. In the sample to be loaded on the gel I have then 140mM Tris, 13% glycerol, 3.75% SDS and 80mM DTT.
Do you think there is maybe something I can optimize to prevent proteolysis? According to me under such conditions proteases should be 99% killed LOL
Thx! :- )
AleX
I recently started doing some proteomics and right now I have some troubles in running preparative SDS gels. Indeed, I often find proteolytic products spread through the whole gel.
The protocol I am using now is the following:
I break yeast cells by bead-beating in a lysis buffer similar to the sample buffer containing:
0.1M Tris pH 7.5
10% glycerol
Protease inhibitors (Roche)
1mM PMSF
2.5% SDS
Before loading the extract on a gel I just add sample buffer and heat the mix at 95°C for 5’. In the sample to be loaded on the gel I have then 140mM Tris, 13% glycerol, 3.75% SDS and 80mM DTT.
Do you think there is maybe something I can optimize to prevent proteolysis? According to me under such conditions proteases should be 99% killed LOL
Thx! :- )
AleX
use powerLaemmli which is sample buffer as suggested plus protease inhibitors; however, the most critical step in proteolysis is not SDS-PAGE but extraction/lysis procedure;
use powerLaemmli which is sample buffer as suggested plus protease inhibitors; however, the most critical step in proteolysis is not SDS-PAGE but extraction/lysis procedure;
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Hi!
I am agree with you... that's why I lyse cells in a buffer which is nearly the same as sample buffer (2.5% SDS, 10% glycerol). The only difference is that I do not have DTT and bromophenol blue (for quantitation) and I use pH 7.5 instead of 6.8. However in spite of this I still see some proteins which are smearing...
But probably I cannot completely avoid proteolysis at 100% (and sometimes with such "super mass spec" just a small amount of protein can be easily detected LOL)
At which protein concentration would you store the extracted proteins? Would you maybe boil them to 95°C before storing or just heat when I need to load them on a gel?
Finally I calculated that, since I add sample buffer before SDS-PAGE to my lysate (which already contains SDS, glycerol and Tris), I have at the end 13% glycerol and 3.75% SDS when I load the proteins on the gel... Do you think that I have too much glycerol?
Thanks a lot!!!!
I guess you lyse your cells a 4 celsius. But if not, there may be your problem!
Being that you're adding SDS directly to your lysis buffer, I assume that you are not using these extracts for anything other than the western. If that is the case, then you can try a much simpler total yeast protein extraction. Spin down your yeast cells and add an equal volume of 2x SDS buffer loading buffer + 80 mM DTT. vortex it briefly to resuspend the cells, then freeze it in liquid nitrogen. Then immediately transfer your frozen sample to a boiling water bath. Boil 5 min, spin down the junk, and load your supernatent on the gel.
[I am agree with you... that's why I lyse cells in a buffer which is nearly the same as sample buffer (2.5% SDS, 10% glycerol). The only difference is that I do not have DTT and bromophenol blue (for quantitation) and I use pH 7.5 instead of 6.8. However in spite of this I still see some proteins which are smearing...
But probably I cannot completely avoid proteolysis at 100% (and sometimes with such "super mass spec" just a small amount of protein can be easily detected LOL)
At which protein concentration would you store the extracted proteins? Would you maybe boil them to 95°C before storing or just heat when I need to load them on a gel?
Finally I calculated that, since I add sample buffer before SDS-PAGE to my lysate (which already contains SDS, glycerol and Tris), I have at the end 13% glycerol and 3.75% SDS when I load the proteins on the gel... Do you think that I have too much glycerol?
Thanks a lot!!!!
[/quote]
in general, it is not recommended to store proteins in sample buffer as resolution in SDS-PAGE is lower than with fresh prepared samples;
more than 5% glycerol is superfluous or even risky as probes become to heavy and may pour down between gel and plate!
HI guys
I have a little querry.
I have got a protocol where PBS is used in cells washing and urea, thiourea, chaps and Tris (pH 8.0) in lysis buffer for lysis. Also I have got another protocol of western blot of proteins where its mentioned TBS/TBST (pH 7.5-8.0) for wahing and probing of anitbodies.
I was just wondering if the PBS for washing and TBS/TBST for western would interfere with determination of a protein.
Could anybody give me the idea.
I have also querry if the pH of the TBS/TBST should be same as that of lysis buffer used.
Thanks