I am seeing high gelatinase activity at site between 55 and 35 kDa and unable to find out the reason for this? Samples are MDA-MB-231 cell lysates and spent media.
Lane 1: protein ladder
Lanes 2 and 3: cell lysates
Lanes 4 and 5: spent media
Has anyone else seen gelatinase activity at approx 40-45 kDa size? Woudl really appreciate any insight.
Some of the non-gelatinase, low molecular weight mmps will show up on gelatin at high concentration. See attached paper.Chapter Zymography as a Research Tool in the Study of Matrix Metallo...
However, in the 50-65 kDa range you are seeing processing products of Mmp1 and other gelatinases.Article Matrix metalloproteinase production in regenerating axolotl ...
if you see more of a smear of digested gelatin, you have bacterial contamination enzymes.
Ellen A G Chernoff Thank you Prof. Chernoff. It possibly is MMP-1. Thank you again for sharing.
This can also result from the breakdown of gelatinases during sample preparation; the catalytic domains of either MMP2 or 9 alone will exhibit gelatinolytic activity on zymography gels, so if the intact molecules have been cleaved (or just mechanically sheared) in the hinge region, you'll get lower MW bands on a zymogram.
Bryan Douglas Crawford Thank you for your insight. Any ways to avoid that? I have my samples (spent media) on ice throughout.
Are you adding protease inhibitors? It's also important to avoid shearing and/or freeze-thaw cycles (that will definitely cause the sort of breakdown I think is causing this). But it may be unavoidable if you're using conditioned media as your starting material, as the hydrolysis of the proteins in the media is happening before you're even collecting it.
Bryan Douglas Crawford I am not adding any protease inhibitors because I do not want the gelatinases to get inhibited. I do not do freeze thaw as I use the samples straight after harvesting.
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