Hi,

I use 10% gelatin gels for MMP2/9 activity assay and I use culture medium as a sample. The problem is that the samples are not running in the lower gel but instead, stack to the border between upper and lower gels or run only for 1cm in lower gel. This happends also in ready gels, so the problems may be dealing with the samples and/or running buffer. I use to run the gel for 3h in 20mA and the gelatinolytic activity appears only in the border between the gels. However, the standards run normally. Anyone having ideas to solve the problem?

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I think your culture medium may contain some cell debris.
Try spinning the culture medium in high speed for 5 min, then load the supernatant onto the gel.

I hope this may help.

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Thanks for advise! unfortunately, that didn´t solve the problem. I´ve also used several different samples e.g lysates and media. Could there be something wrong with the running buffer? I´ve also wondered, is SDS bound to gelatin in the gel and if it´s, how can it bind the samples? Should I use more SDS? The binding of SDS is crucial for the proteins moving in the gel giving a negative charge to proteins.

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Did you use loading buffer (sample buffer) containing SDS? if not the MMP2/MMP9 will start digesting the gelain while the gel is running and may stuck on the top of the gel.

I hope this may help.

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