Hi, all. I am trying protease zymogram with 12.5% tris-glycine-gelatin gel, the concentration of gelatin is about 0.1%, the gel was run at 130 V, removed SDS, incubated at 37 degrees overnight and then stained by coomassie G250. The proteases were secreted by fungus in the medium with mucin. I attached the photo of the gel. Any one has ideas what should I do to make progress. Many thanks.
Hi Han
Although zymography is a great techniques for protease activity demonstration after SDS-PAGE, however much care should be taken during the whole experiment. It looks from your gel image that you have smeared zymogram bands, which is expected when you used the substrate (gelatin) within the gel. In this case during the gel electrophoresis the active protease will digest the protein and thus leads to the such result. In this case you either should have to control the experiment conditions such as: the percentage of the SDS, the amount of the protease you have load to the gel, and the temperature during electrophoresis run. I advise you to try post-gel zymography using either gelation or casein as substrates.
Hi, Isam, thanks for your response. Can I ask what is post-gel zymography?
Hi Han
Post gel zymography is reacting the enzyme with the substrate after SDS-PAGE. In this case you need to run SDS-PAGE as usual and after you wash the gel to remove or decrease the concentration of SDS, the gel should be soaked in the buffered substrate at 4C for about 1 h to allow the substrate diffuse to the gel at low temperature. After that you need to move them to 37C and incubate for 30 min-1h for reaction. There after you can stain the gel with CBB. I have used this protocol many time before and it gives a satisfactory results. I attached the paper that i used this protocol in it as well as the original paper that described this protocol.
Hi, Isam, thanks so much. My proteases have high activity and would break down other proteins during normal SDS-page, so I have to add proteaes inhibitor when prepare the loading samples. Do you think the inhibitor will effect the post-gel zymography?
Dear Han
Although i do not understand what you mean by degrade other proteins during SDS PAGE, I do think this will not affect the the post-gel zymography. Also the second paper that i sent you also used both active enzyme and that inhibited by protease inhibitor, which will be of great help in your case. Did you know the type of your proteases (is it Aspartic, serine, cystaine, or metalo-protease)?. Also do you use crude enzyme preparation of purified one?. Did you add the protease inhibtor to avoid degradation of other protein or to stop auto-digestion of your proteases them self?. In my case my protease has autodigestion in SDS-PAGE buffer and I increase the concentration of SDS in the sample buffer to stop this auolysis during SDS-PAGE.
Good luck and if you have more questions your are most welcome
Hi Callie,
Do you run the gel at room temperature? If yes, then try to run your gel at 4 oC in cold room or in refrigerator, it might help you. In my case this method helped me. If you tried this also then go for post-gel-zymography as suggested by Isam. If you want to add protease inhibitors then you should know the type of your protease, then about reversible inhibitors and a proper method to remove it from gel after electrophoresis. Better go for literature.
Hi Callie, I think there is nothing wrong with your zymogram. Obviously, your proteinase in the 2 lanes is actively chewing up the gelatin during its run. As others suggested, you could try loading less sample and/or run the gel at 4oC.
My main concern is the band of lysis at the bottom of the gel. You have some spill-over into adjacent lanes when you loaded the samples, and it seem that you have a very active proteinase that is of very low MW. What is the MW of the last standard?
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