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-   -   band shift in control lane! (http://www.molecularstation.com/forum/electrophoretic-mobility-shift-assay-forum/83152-band-shift-control-lane.html)

deepapmb 07-14-2011 01:40 PM

band shift in control lane!
 
2 Attachment(s)
Hi all,
I am currently doing EMSA with pierce light shift EMSA kit. For binding reaction I use 20 pmol of biotin labeled DNA, 5 microgram of protein (his-tagged protein), hundred molar excess of unlabelled DNA , 50 ng of poly dIdC and the 1X binding buffer to the final reaction volume f 20 microlitre. I incubate it at room temperature for 30 min. After that I run it in 10% PAGE gel at 100 Volt ane then transferred to nylon memebrane with 0.5X TBE buffer and use that membrane for detection. First time when I did , I got good band shift in all the lanes which contain protein. Now in the repeatation experiment , I am getting a band shift in the control lane also which contains only the biotin labeled DNA WITHOUT any protein. I am attaching my blot here . What could be the possible reason for the band shift. Is it a non specific binding , or any protein contamination in my reagents. For the repeatatiopn experiment I used the same ).0.5X TBE buffer which I used for the first experiment. Please help.

Thanks in advance for your replies

deepapmb

mmorgan 07-14-2011 07:26 PM

Re: band shift in control lane!
 
Hi, It's hard to tell from the images, but it looks like the shift in mobility is very small. I wonder if it might be a problem with the probe itself (since you see it in the lane with only probe and no protein).

Do you polyacrylamide-gel purify the probe before (or after) labelling to ensure that it is homogenous. Perhaps the upper band is not a gel shift, but rather its a larger piece of DNA that is in your probe sample.

You could just run out some probe on a acrylamide gel without any EMSA binding buffer and see if you still see two bands. If so it's definitely something in the probe itself.


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