Protocol Online logo
Top : Forum Archives: : Molecular Biology

Strange background in lanes using Pierce Lightshift EMSA - (Dec/19/2007 )


I seem to have background in my sample lanes in my EMSA (see attached pic). there are seven samples shown on the gel, the last spot on the gel at the far right is the control EBNA biotin labelled DNA that comes in the kit (in only ddh20 and loading buffer). The other six bands are: in lane 1) Biotin labelled DNA 2) Biotin labelled DNA + protein 3) Biotin Labelled DNA, protein and competitor. lanes 4-6 are the same, but with mutated DNA. The only reagents that are constant in all lanes (excluding the EBNA control) are:

Binding Buffer
poly dI-dC
Biotin Labelled DNA
Loading buffer

there are no additives other than the above in all lanes. I can assume that the loading buffer and h20 is not responsible as the EBNA DNA lane does not have the background. Could anyone please give any suggestions why this is happening?

Thanks in advance



well, I can offer two suggestions that may help.

first, it appears that your primers and/or protein samples are degraded. degradation is often the problem when you get a low-MW smear. I suspect the problem lies in the DNA (even though not all wells are the same, the first lane is smeared and it only got DNA)

second, I think it would be good to either run the gel a bit more slowly, increase your % of acrylamide, maybe reduce your loading buffer glycerol content? the arc over your sample lanes is not so great, and even if the probes weren't smeary it would be tough to get useful data.

so, the lack of shift from this experiment would not concern me. if you clean up the other issues, you might get the shift you're looking for.

good luck!