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supershift of gel shift assay

supershift of gel shift assay - Protocols and Methods Forum

supershift of gel shift assay - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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Old 12-10-2003, 11:05 AM
Takeshi Miura
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Default supershift of gel shift assay



Nice to meet you every one!!

Sameone mentioned protocol about gel shift assay.
I can see specific DNA-protein complex band in gel shift assay.
And, i want to identify the protein binding oligo DNAprobe.
Now, i expect it is one protein. So, now try supershift assay using
this protein specific antibody.
But, no any change between control(no antibody) lane and supershift lane.
my protocol about super shift reaction is...

protein extract 2ug and PolyL etc.
30min on ice
add antibody
30min on ice
aplly, electrophoresis using 8% PAGE
and detection by DIG system(non-RI system)

After added antibody, 30min reaction time is enough?
on ice is adequat?
if depend on type of protein, please teach me about your good protocols!
Thank you!!



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