The sample was obtained from protozoan cell free extract (secretome) and subsequently concentrated by ultra-filtration. The problem is that after running the gel, the marker (Spectra Multicolor) gets stuck towards the top of the resolving gel and so does the samples (~10μg) half way through.
It's a 10% acrylamide gel with gelatin substrate (1mg/ml), run at 100V and coomassie stained. I have run them earlier under same conditions with no issues, but now I keep on getting this problem. All the buffers are freshly prepared. Any idea/suggestions?
(attached pictures of both zymogram and conventional, samples run simultaneously)
Are you sure there was no error in the bis-acrylamide crosslinker concentration?
Doesn't seem so, because the same samples run simultaneously on conventional gel (without gelatin substrate), the marker and samples migrate through. I just attached the picture above.
Hi Rounik, I assume you are using the same gels-either bought or home made.
Streaks in zymograms are not uncommon if you haven't filtered the samples before loading or if the SDS concentration in the gel or sample buffer is too low.
What got my attention is that the upper gel has a zone of lysis at the top of the MW markers.
To me, that looks like spillover from the other lane. If the MW standards have BME, then that would affect the activity of the adjacent lane. Conversely, a spillover from the adjacent sample lane that did not have enough SDS would stop the MW markers in their tracks-if the gel did not have SDS already in it.
Try separating the MW standards by 2 lanes from the samples to see is spillover is the problem.
Regarding the white bands in the upper zone, sometimes happens also to me even without spillover from adjacent wells (I call it "comb effect"), and it seems like gelatin started the migration. Instead, I'm a bit concerned about the presence of a blue band in the combs in the second gel (and you can see it a little bit also in the zymogram) which make me think about the presence of aggregates, so there might be the chance that the amount of SDS is not enough or the proteins just denature during the concentration step. Also, in the second gel there are not clear bands of proteins but smears, which can suggest the presence of detergents (but if it is a secretome, I don't think so) or more likely degradation of the sample.
Regarding the MW marker, I can't be of very help since usually I load positive controls to see the gelatinolytic bands rather than molecular weights, but the times I tried this never happened...Did you put your MW marker with DTT/B-MSH and boiled it prior to load? This is essential!
Just to follow or on Alessadro's observation, if you make your own gelatin gel zymograms, the gelatin should be salt-free.
Any salt in the gel will distort the run.
@Steingrimur ---The samples were filtered trough and the SDS is 10% which I used before. I don't think its a spill over from another lane. I watch them very carefully as I load, usually one could see a spill over if one looks at it minutely while loading (happened to me in past). Yes I make all these gels. Regarding the salt-free gelatin, I have some spin columns but I rather keep them for my other samples, do you know of any inexpensive protocol to do this? maybe some ticks that I don`t know :)
@Alessandro ---Regarding the presence of aggregates, could it be another 'comb effect' because I ran my other samples they don't seem to have this (picture attached). Even if it does, any idea how much SDS shall I use? And as for degradation of the samples, could it be the proteases are degrading my sample? About the MW marker, in the product sheet it says not to boil it.
Ok perfect. I think that it is really astonishing that the MW marker in the zymogram is not running while in the normal SDS page it runs perfectly, considering that the only difference is the gelatin. Regarding the degradation of the samples, it could be due to proteases. I don't know if you are interested in specific gelatin-degrading proteases. If so, you can add inhibitors but being careful not to inhibit your proteases (e.g. if you add EDTA the metalloproteases will disappear)!
Regarding gelatin (from pig skin, I don't remamber the stokes), usually I dissolve the powder in 1.5M Tris-HCl pH 8.8, warm until it is dissolved and replace a part of the Tris used in the running gel of the SDS-page with the gelatin. It always worked for me. You can also dissolve the gelatin in water and replace a part of water in the runnig gel.
Finally, for the MW marker did you load a higher amount in the zymogram than in the SDS page? Otherwise your MW marker will not be visible..
Hi Rounik, I think Alessandro might have the solution to your problem.
You make your own SDS-PAGE gels and zymograms.
For your SDS-PAGE, the markers run nicely. For your zymograms, they do not run at all.
As I see it, the problem might be the gelatin that you use is full of salt, which retards the run.
Salt free gelatins available for zymograms:
http://www.sigmaaldrich.com/catalog/product/sigma/g8150?lang=en®ion=US
If you don't want to buy gelatin for zymograms, you could dissolve your gelatin at 10mg/ml in 0.1M acetic acid. If it doesn't dissolve, add HCl to it until it is dissolved. Put that dissolved gelatin into a dialysis bag and dialyze against 0.01 M acetic acid. If you use 0.1mg/ml of that gelatin in your zymograms, you should not have a salt problem.
Yes that was precisely my reasoning, the only difference is the gelatin, so it must be that. It worked now, I desalted the gelatin and changed the concentration to 0.4mg/ml. Attached the gel from Sunday, I did not use my samples this time, only the MW. I loaded the same amount of MW as in the SDS page.
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