Hi everybody, I must do gelatin zymography but my samples don’t show any activity. I use a serum control to show the lysis bands of MMP 2 and 9. That control shows lysis activity but no the other ones .
Whats my problem? I use a non reducing sample buffer and I suppose that all my protocol is ok so that serum show lysis bands.
Hi there,
The basic explanation is that there is no POI in your sample or it is inactive for some reasons (inactive from the start or unable to recover activity after electrophoresis).
You could try to do a protease assay directly with your sample, without electrophoresis. If you see nothing, there is no point in electrophoresis, otherwise you could work on optimising your technique.
There are non-specific protease assays like the azocasein method (http://www.jbc.org/content/171/2/501.full.pdf, recently elaborated again in 10.1155/2016/8409183), several companies now flog substrates that work on this principle, but use fluorescent dyes for greater sensitivity.
Hi,
There could be 2 reasons for no proteases activity. First, your other samples do not have MMP activity or relatively in very low concentrations which is not detectable. If its in low concentrations, may be you can try silver staining instead of coomassie staining. Second, the MMPs in other samples are in inactive forms. Probably you can add activating agents such as APMA to activate MMPs activity.
Regards,
Harleen
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