Proteins samples not migrating from stacking into resolving gel - (Aug/25/2006 )

I use a 3.5% stacking gel and a 7.5% resolving gel, yesterday I ran a gel and stained it with coomassie blue. The protein had left the wells but not migrated into the resolving gel. Is there any explanation for this?

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Please write down the exact composition of your gel solutions. Then we can have a better look.

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think of detachment of phases and air between upper and lower gel; or wrong mixture of the gels such as wrong pH which means wrong concentration of Cl-? maybe strong aggregation of proteins? (depends on proteins and preparation for SDS-PAGE)

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Resolving gel I use - 4.85mls deionised water, 2.5mls 1.5M Tris-HCl pH 8.8, 100ul 10% SDS, 2.5mls Bis/acrylamide 30% solution, 50ul 10% APS and 5ul Temed

Stacking gel - 6mls dH2O, 100ul 10% SDS, 2.52ml 0.5M Tris-HCl pH 6.8, 10ul Temed and 50ul 10% APS and 1.32mls bis/acrylamide 30% solution

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I think that your percentajes are a little low I use 15% per resolving gel and 4% for stacking gel. Try this percentajes

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the percentages are not likely to be your problem, in my opinion

I would verify the pH of your tris buffers first, that is the easiest to fix

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agree with aimikins. check buffers. what about the sample buffer, is that good? and did you remember to add sds in your sample buffer?

- viv

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