Probe labelling foe gel shifts - (Aug/09/2006 )
I am doing Gel shifts using the non-radioactive DIG kit from Roche.
The control proble labels up much more efficiently than my test probes (they are the same size). How do I improve probe labelling? The kit recommends using HPLC purified oligo's. The oligos I am using (my probe is annealed oligos) are just desalted. Is it critical to use HPLC purified oligos?
I have biotin-labelled standard desalted oligos without a problem using a kit from Pierce
in their literature that comes with the kit, they say that sometimes oligos just don't label well. one possibiliy is secondary structure (does your oligo make a hairpin or dimers with itself?)...and sometimes it just doesn't work for no real reason, probably just that combaination of bases is not friendly to the transferase.
so, I would check your primers for secondary structure?
My probe is labelling well now and I'm seeing a shift (smeary) when my purified protein is present. I also saw this clearly just by staining the gel with EtBR but wanted to go on with the labelling to do competition experiments.
Something weird is happening though with my competition. When I add excess (125 fold) unlabelled probe - I seen to get a better shift! I know that my protein binds specifically to this sequence (the sequence I am using is 29 bp) as I've tried to bind it to a random seqeunce and seen no shift.
I will proceed to do more expts with more unlabelled probe (500 fold excess) but this is really confusing me! What do you think might be happening?