supershift-EMSA problem - (Mar/31/2006 )

Hi,
I have problem with my supershift experiments. I am getting the supershifted band but it is not followed by either desappearance or reduction of any of the DNA/protein complexes (they sometimes even intensify). I use anti Sp1 and anti MAZ Abs which are known to work quite well.
Does anybody know is this publishable or why this kind of thing happens?
All the help will be greatly appreciated

Grooya

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Hi, I think that you should try reducing the amount/radioactivity of the probe you put in in the assay and/or increase the amount of antibody used for the super shift. You may be adding too much probe, another possibility is also adding too much proteins. Let me know, if you try.

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thanks for the tip. I already tried to increase the amount of Ab but it only intensified shifted complexes. I use 1,25 - 2,5 micrograms of protein and 0,5 ng of poorly labeled probe (14 000 cpm).

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I'm using 0.02 micromolar probe (10 0000 cpm) in the reaction, that represent 0.02 ng of probe! My oligonucleotides are labeled by end-filling with Klenow and 32P dATP (3000Ci/mmol). The oligo are synthesized to have 5'-protuding ends after annealing. I routinely get about 80 000 cpm per microliter of purified probe. So I really think you should try getting a better labeling method to be able to reduce the amount of probe.

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