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problems with second dimension


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#1 duncan

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Posted 11 December 2001 - 01:29 AM

Hi

Our lab is having some problems with 2nd dimension SDS PAGs. Silver staining of our gels reveals that the proteins are well separated at the top half of the gel. However, they are stacking up at along a mysterious wavy interface before they reach the bottom of the gel. The BPB dye migrates normally, though it is a little diffuse. The gels appear to be set properly and are robust in the staining procedure.

Some people have suggested that a 10x running buffer stock may be the culprit (contains SDS). Others have pointed at a 1.5M tris bottle. Yet another person suggested that the gels were set too fast.

I have had the same problem with 10 or 12% gels cast in a chamber or singly.

Any ideas?

Thanks


#2 Pascale

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Posted 14 December 2001 - 05:52 AM

Hi,
This might sound weird, but how old is your acrylamide? I seem to remember that, long time ago, I had this kind of unresolved bands at the bottom of sequencing gels, in the bottom quarter or third of the gels, depending on the length of the electrophoresis. All theses desappeared with a fresh bottle of acrylamide.

#3 duncan

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Posted 14 December 2001 - 08:03 AM

Problem solved.

The gel tank buffer contained 10-fold too little SDS.  This makes sense since the SDS migrates through the gel and it was not replenished by the buffer.  So, eventually the SDS dissociated from the proteins and  they stopped migrating.

(Edited by duncan at 6:04 pm on Dec. 14, 2000)


#4 franck

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Posted 13 March 2002 - 01:27 AM

did you use a pre-run of 15 min ? this is like a stacking gel, but perso i prefer bleu colloidal coloration

#5 kaiya

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Posted 23 May 2002 - 08:21 AM

I am having a similar problem.  May I ask how you make your SDS-running buffer?  

#6 Sciad

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Posted 26 June 2002 - 07:13 AM

Are you running a gradient gel? If so, you may want to check whether the solution comprising the lower fraction of the gel has a problem with polymerisation (too fast??). This would explain why the upper portion is o.k.




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