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Position: Discussion forums > Bioscience biotechniques discussion forums > Proteomics and protein biochemistry forum
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Protein will not enter 5% nondenaturing gel.
Posted by: winquist79 (IP Hidden, New member, 1)
Date: August 8, 2005 08:30AM
I'm trying to put together a bandshift assay with a regulatory protein and a promoter sequence that is well documented to be regulated. The DNA-sequence has multiple binding sites for the protein and is 3' labelled with digoxygenin. The problem is that neither free DNA nor DNA/protein complex can be detected after blotting and imunofluorecense evaluation. Tries with a promoter sequence with fewer binding sites have been made, with a detection of free DNA in the gel as a result. To solve this problem, I have used different salt concentrations and different compositions of the gel (3, 4 and 5% polyacrylamide gel).
Anyone got any suggestions where I can continue to look for an answer?
Re: Protein will not enter 5% nondenaturing gel.
Posted by: shobhit (IP Hidden, Unregistered user, )
Date: August 9, 2005 12:30AM
u can adjust dna : protein concentration by doing a kind of titration of protein conc. range versus constant dna , say 5ng in each reaction against different protein conc., on ice for say 30 min.
addition sequel should be : buffer, add DNA, keep on ice for 10 min., add protein , incubate for further 30 min., stop with DNA loading dye and load onto 5% native PAGE (prerun at 4degree at 160V). for non specific biding, i have used polydI.dC in these reactions ........around 100ng per 20ul reactions. try these !
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