I am facing some difficulties with caesin zymogram assaying the activity of MMP-3 and -7 from human samples. I am not getting any activity, but the proteins are there in the western blots. After running, some protein clumps are seen near the wells. I also tried the same with altering the SDS percentage, but it didn't work. Can anybody suggest a way out?
I would switch to gelatin gels because these MMPs are not very efficient on common proteinase substrates.
MMP-3 & -7 are not gelatinases. MMP-2, -9 and -13 can be assayed in gelatin gels and the protocol is pretty standardized.
So, gelatin will not work in this case I guess !
Hi, gelatin will work, I just suggested it because gelatin is usually an easier sustrate to cleave-it is already denatured.
As for the zymogram, are your markers running OK? Are your samples APMA treated? Do you have a positive control lane (e.g. trypsin)?
Sir, Thanks for your valuable suggestion. But as far as I know, gelatin is not a good substrate for Matrix metalloproteinase 3 and 10. If Gelatin is added in SDS page as substrate, MMP-9 and MMP-2 will give bands, not 3 and 10.
Yup, my markers are running good. I cant get the abbreviation APMA...will you please tell me? I had positive control with BSA.
Hi Anirban. You are correct, there seems to be more people doing casein gels of stromelysin than gelatin gels.
APMA is aminophenyl mercuric acetate that has been shown to activate some MMPs
http://www.jbc.org/content/268/21/15435.full.pdf
I was mentioning trypsin as a positive control for caseinolytic activity. Just to check if the run or the TX-100 washes were OK.
Lastly, You asked for a reprint of one of my papers. Could you give me your email address so I can send it to you?
Thanks.
Keep few things in mind before you do SDS Denaturing-Renaturing zymography. make sure you do not treat your protease too harshly in SDS sample buffer before loading. 30 min treatment at 37 deg C is suffice to coat it with SDS without permanently inactivating it. After electrophoresis make sure you completely remove SDS by incubating the gel in ~15-20% isopropanol solution to renature protein.
If your Casein substrate contains too much calcium, it may interfere with zymography as calcium makes casein difficult to denature. Proteases work best on denatured substrates. You can either try dye-labelled caseins or hemoglobin. When dissolving protein substrate use 1-2 M urea to denature and then dilute.
Firstly, thanks both of you for the valued responses.
@ Dr. Stefansson, I ll try my samples with APMA definitely and shall let you know the results.
My e-mail address: [email protected]
@ Dr. Siddiqui,
We usually dont treat our samples with SDS separately, when we add sample loading dye with our sample, we add a little SDS within it. After electrophoresis, we remove SDS with 2.5% Triton X -100. After washing we usually incubate the gel with a buffer which contains tris HCl, brij, and Cacl2. I ll try my incubation buffer without CaCl2 and shall get back to you. Thanks.
Thanks and Best Regards,
Anirban
Even Zncl2 will help you as MMPs require Ca n Zn for their activity.. Also the samples should be as fresh as possible and if need to be stored, store them at -70 degrees.. overnight incubation with tris, pH 7.4, triton, Zncl2 at 37 degrees should also help you with the activity.. all the best
I had a student who was having trouble like this, and it turned out she was using standard SDS-PAGE sample buffer, which included DTT as a reducing agent. Obviously, you need to use a non-reducing sample buffer to get the enzymes to refold correctly after electrophoresis.
Cheers
I hv tried using DTT with my samples, even in two different concentrations. Will you pls suggest what will be my DTT concentration? Usually I add 5 ul of sample loading dye with 2 ul of my sample (human serum).
Yes, you need to use non-reducing sample buffer. No DTT... no beta-mercaptoethanol.
If you've had DTT in your sample buffer, that's why it's not working.
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