I am studying MMP-2 and MMP-9 activity by gelatin zymography. The bands I have obtained are smeared up (within their lanes). Can anyone suggest possible reasons for this?
can you post a picture of the result to have a more clear idea on how it look?
Cheers,
Giulio
We loaded 4 samples. We were just optimizing the setup. The bubbles probably you will see are after we transferred the get for scanning. Another interesting thing happened, when we were using the sample buffer we could not observe any bands. This smeared up thing happened when we loaded our samples without the buffer. I would just like to know such a thing can be result of exactly what? We homogenized our cardiac tissue sample with an extracting buffer. After performing bradford, we loaded the supernatant to the wells. Can there be a possible issue with gel composition or we loaded too little or too much of protein?
Hello,
Some experts say that it is due to distilled water.
Good luck
Hi Sulail,
That looks like an overloaded gel to me. Other things to consider: use a larger stacking gel, run your gel more slowly, and try running less sample and developing longer.
Cheers
To add to Dr Crawford's comment - run your gel slowly, which reduces smear and means it doesn't run too hot. Timing of and gel incubation temperature are important. I don't think you tell us what your samples are, and how you have prepared them? This may influence the activity you can see. Have you a positive control where you are confident you have activity to test your protocol out? My only other suggestion is on your gel loading - just run a small series of samples at different dilutions / concentrations, to optimise. good luck.
Thank you Dr. Riley for your worthy comment. Our samples were cardiac tissues. We crushed and homogenized 30mg in 100 ul extraction buffer (SDS, mercaptoethanol, glycerol and bromophenol blue). We used protease inhibitor (EDTA free). We loaded 50 ug of the protein.
The first thing i would like to ask, are you sure you want to add protease inhibitor in gelatine zymography, where you want to detect protease activity?
the second thing is the loading. We usually load 1µg or 2µg on gelatine zymogram. 50µg seems too much loading.
Dear Richard, EDTA free protease inhibitors are ok for gelatin zymograms in order to detect MMP.
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