I am purifying a single strand DNA binding protein complex. It was binding to radiolabelled oligos and was giving shifts in agarose and acrylamide gel. Suddenly now it stopped binding. I am following the same purification and binding protocol. I tried making all fresh reagents and oligo, but no help. Has anybody ever faced a problem like that?
Dear Anukana,
One reason could be that your oligos might undergo dimerization upon long storage. so, be confirmed that whether your oligos are single stranded. Second point is that inclusion of Mg++ ions, if it is insufficient, it could lead to failure in binding.
All the best wishes,
Regards
Rajkumar
try to heat up your oligos to 80 C for 15 min and cool down quickly in ice before using. that should prevent self-dimerization of the oligos.
Hi Anukana,
is the binding mode of your protein specific for a given sequence or just for single stranded DNA? In many DNA protein complexes, the contribution of non specific electrostatic interactions can be important. If it is case for your complex I would suggest checking the pH and ionic strength to make sure that the non specific interactions can take place.
Have you characterized your protein of interest that binds to ssDNA? And how did u made sure that the same type of protein have been extracted in the subsequent trails?
Hi it is important that you do not freeze-thaw your protein as after several cycles of this procedure it will be degraded fully. Always use fresh protein aliquots and discard each one after a single use. Do you use fresh protein aliquots normally?
Max.
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