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|Electrophoretic Mobility Shift Assay Forum EMSA Forum. Discuss the Electrophoretic Mobility Shift Assay protocols or technique and the assessment of DNA and RNA binding proteins here. Also post questions and troubleshooting about the shift assay.|
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band shift in control lane!
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
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.
|band , control , lane , shift|
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