I am doing non-radioactive EMSA with biotinylated oligo. My oligo is GC-rich in sequence. I used to get retarded band in my gel which was successfully competed by cold chase. Other experiments suggested the binding of Sp1/Sp3 to that region of oligo. But i am not getting any supershift in my gel with Sp1/Sp3 antibody. Shifted bands are very heavy and in most of the case stucks near the well. what modification i should do in my experiment. I am following Pierce EMSA kit manuals for my experiment. Any specific criteria for selecting antibodies for their use for supershift. When to add antibody in binding mixture of oligo and nuclear lysate. If anyone has done the supershift with Sp1/Sp3 earlier then please share the protocol and any troubleshoots.
Supershift is sometimes tricky - antibody can affect probe-protein formation. Did you try various competitors to track the exact site of binding? I am using set of unlabeled competitors - each with one or two mismatches (when compared to the probe).
Alternatively, think about another factor - eg Egr1 often competes with Sp1.
Thanks Martin, for your valuable suggestion. Actually, i have used site mutated probe for competition and i know the exact site of binding as mutation of that site unable to compete with binding. I have performed the experiment with egr-1 siRNA as well as Sp1/Sp3 siRNA and i got the involvement of Sp1/Sp3 but not egr-1. other experiments as well as inhibitor of Sp1, mithramycin also confirmed the involvement of Sp1/Sp3. But again supershift is not working in EMSA exp. Do you suggest, what should be the sequence and incubation of adding components in the binding mixture.
I have tried adding antibody at various times (together with extract, later with Bi-labeled probe or just before loading - results were the same. But not for Sp1, I have never observed supershift with Sp1 antibody.
You can try WEMSA (ie EMSA binding reaction and elfo separation with subsequent Western blotting - typically requires much more (at least 10x?) protein+probe than EMSA). Can be difficult to load on the gel.
The other possibility would be performing large scale (10-100x) EMSA binding reaction, add streptavidin coated beads (magnetic or cellulose) and pull down trapped factors. Wash the beads, boil in SDS loading buffer and run Western blot.
Later experiments (WEMSA and DAPA), i have done and i got the desired result. With those supportive data i submitted my paper but now reviewers asked for supershift in the EMSA gel itself. I am still trying for the supershift. Can you suggest if adding less or more nuclear protein in the binding reaction will help. Next, how long and at what voltage or mA i should run the EMSA gel for getting supershift band as my retarted band is very heavy and difficult to come down from the well.
Wow, I can not believe, reviewers insist on supershift, provided you have such evidence!
You can try to titrate both antibody and protein concentration, however, supershift should be done at rouhly same conditions as EMSA to be useful as evidence. Try low concentrated gel (4-5%), lower the amount of bisacrylamide. I would keem voltage within normal range (eg 100V ) for prolonged period - even several hours, rather than increasing V for shorter time. Cool your gel and buffer during separation - for prolonged runs, you can change the buffer.
I suppose you have already done it, just in case - make sure, your Sp1 antibody reacts with Sp1/3 under native conditions.
I ll keep my fingers crossed for you - I would try to persuade reviewers rather than Sp1&Co. It is known, that transcription factors often work as a part of huge complex of proteins. Those can prevent antibody from reaching Sp1.
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