Hi,

I have a new problem with my SDS gel. All of my proteins come down to the bottom of gel! My buffers PH are correct, but I don't know what can be the reason? What's your opinion and does anybody have any experience like this?

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Do you have an image for us?

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It sounds to me like you have protease contamination, and all your samples are being degraded into tiny little bits.

Ginger

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what is the size of your protein and the concentration of acrylamide in your gel?

are you sure it is polymerized completely?

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Hi; Thanks for your replies. I'm sure about my lysis buffer as the other people are working with that and also I have worked with the other people samples and the problem still exists!

I have tried 10 and 12 percent gel and it polymerised very good. I don't know what's the problem?! but every protein is at about 16 KD!

What do you think about that?

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How does your ladder look?

Ginger

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R u running a mini gel or maxi gel apparatus?

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Do you use fresh protease inhibitors? Perhaps you could also raise the amount of it just to be sure that this is not the problem.

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The ladder runs fine(without any problem!);
Its a minigel,
And I use fresh protease inhibitor.

Its so strange as I had been done a lot of western before; but now I don't know what's the problem!

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are you staining the gel or only blotting?

how does the stained gel look?

maybe you are seeing some non-specific staining on your blot.

try a gradient gel, 10-20%.

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