I am trying to detect MMP2 activity through gelatin zymography. I do can see clear bands. But all my bands stuck in the top of gel. however the ladder passed through...also when i run the gel, the dye of the samples also passed through. I guess there must be some problems when i prepare my samples, could anyone please help me? thanks a lot.
I used Novex 10% gelatin pre-made gel from Thermofisher.
Samples were extracted from kidney tissue with NP-40 lysis buffer. mixed with 2X tris-glycine SDS sample buffer with no heat blotting and reducing agent.
Thanks!
I used following protocol.. and it worked good..!
SDS-PAGE gel 7.5% (containing 0.5mg/ml gelatin). After electrophoresis at 4 degree Celsius, the gels were washed and incubated overnight in 50mM TrisHCl pH 7.5; containing 10mL CaCl2, 1uM ZnCl2, 0.02% (W/V) NaN3and 1% (V/V) Triton X-100. Next day, the gels were stained with 0.1% (W/V) Coomassie Brilliant Blue R-250 and destained in 7% (V/V) acetic acid.
Here is the reference for protocol..
http://www.conscientiabeam.com/pdf-files/med/95/CR-2014-1(2)-33-44.pdf
You can also add a positive control in one lane to check whether it is problem in your sample preparation /gel/ or procedure.
There are also MMP2 ELISA kit available if you want to use that...
What is the percentage of acrylamide does the pre made gel contain? The only reason the bands are stuck at the top would be because the proteins are bigger than what the gel is made for.
Look for gels with suitable % of acrylamide-bisacrylamide or its pretty easy to make one like Bhairavi Vajaria mentioned
Is the sample processing adequate ? Like Sonication ect ?
Hope the issue is resolved
Good Luck !
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