gelatin zymography - faint white clearance over whole gel - zymography (Apr/09/2005 )

Hi,

I am using gelatin zymography 12% mini-gels. I wash my gels for 1h (3*20min, change 3 times) with triton X100, Tris base pH7.5 (fresh). Than I incubate my gel overnight (19h) 30C in Tris X100, tris base pH 7.5, MgCl2, CaCL2. And stain the gels with amido black.

When I first tryed the protocol it worked fine. I loaded trypsin as positive cintrol and my samples (fish mucus samples) and everyting was fine. I saw a clearance at 20kDa (trypsin) and 115kDa for my samples.

But now I have the problem that it is not working.
When I only load my trypsin I get clearance where the trypsin schould be bit this clearance is bigger than I got before and above the 20 kDa I get faint white clearance over the total surface of my gel. It is darker where my standards are loaded.

I think it has something to do with my gelatine.
I dissolve 1g gelatine in 100ml water and heat it on a hotplate untill the gelatine is dissolved. When I left the gelatine to cool down for 1 h this faint white clearance was stronger over my whole gel.
Then I tryed it again but this time I heat my gelatine (80ml) then add 20ml water on top (cool down) and use it emediately. I also tryed to filter it. The results are bether than when I left my gelatine to cool down for 1h but still not usefull.



I make up my gelatine, trypsin stock solution and washing solution fresh. I store my incubation solution in the fridge (4C). schould I make it fresh?

Maybe the problem is not the gelatine but maybe there is a problem with the buffers.

Can someone help me?
I looked on the net for the protocol but they dont give a detailed description of how to prepare the gelatine or how to store the reagents.
Does anyone have anye idea what the problem or can someone give me a ussefull adress of a internetsite.

thanks

Klaartje

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I think the concentration you use to dissolve gelatin is too high, for nature of gelatin, i suggest you better to use low concentration (4 example, 1~5mg/ml) and do not use stocking for gelatin. the gelatin should be fresh prepare.

if any further question, feel free to contact me. my email--yamacoqi@yahoo.ca
good luck

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I solved the problem that I described on the forum ( clearance over whole gel). My gelatine was not dissolved enough. I heat it now slowly and dissolve if fully and let it cool down before I use it.

However I have another problem and I am not able to solve it.

I load trypsin as a positive control and my samples (fish skin mucus). However I have troubles with a horizontal clearance (line) over the whole length of my gel at approx 20kda. First I thought it was trypsin but even without the trypsin I have the line.
I think it has something to do with the triton X100 in the washingsolution. When I wash 2h (3 changes) I get a strong clearance (line at 20kDa)over the length of my gel and 4 protease bands (115kDa, 90, 60, 50kDa) in my samples. However the clearance interferes with my samples so I can not use the gel. When I wash 3x20 min the clearance is much better (not fully gone) and see 1 protease band (115kDa). So I think the longer you wash the more you get clearance at approx 20kDa.
When I washed 3x10 min the clearance was gone (20kDa) but no protease activity was detected in my samples. I think triton x100 interferes with my results (I use 2.5%triton X100 and 50mM Tris HCl pH 7.5 as washing solution). I shake my gels at 4c in the fridge.

I tried everything. the only thing that I think that I could try is use new triton X100 since I incubate the different gels in the same way.So my problem has something to do with the washing step

I use the protocol described in Fast et al. (2002) Comparative Biochemistry and physiology part A 132 645-657. It worked for them.

I don`t know if you have any suggestions. Any help is welcome.

Thanks

Klaartje

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