did you add the unlabel probe after binding of the label probe or at the same time?

is it possible that you have several DNA/protein complex; the largest (box1) being easely displaced by the cold probe (to give more box2).

binding of the anti-tag can make a to big complex that do not enter the gel...

Didier Poncet

Thank you so much!

1, About your first question, I add the unlabeled probe to the reaction mix and incubate 10 min at RT, then the labeled probe.

2, I'm not sure if there will be multiple DNA/protein complexes, the nuclear extract is a mix of many proteins, maybe I will try purified proteins next to avoid this kind of problem.

3, Do you have any alternative antibody suggestions for my supershift experiments except for anti-tag ?

Best,

Kuangxin

looking a second time to your gel : you are in large excess of biotin-probe as a lot of it stayed a the bottom of the gel;

maybe using less cell extract you will shift all the probe unless a fraction of the probe is not functional

as you added the cold probe before (which is the right thing to do) it means that box2 complex is not desplaced by the cold probe= could box2 be the results of a protein binding to the biotin part of the probe?

I would definitively try an EMSA with untransfected cell extracts; this will tell you if the box2 or box1 comples is the one you are looking for.

for the antibodies: the ha antibody does not do a lot to the complexes (could the HA tag be inaxessible? the eGFP antibody slot maybe you have a gel loading pb as there is less free probe in this slot...

Didier Poncet

Thanks a lot!

Good news! I recently got good results by reducing the biotin-probe and nuclear extract, also I doubled the cold probe input, it seems like the excess amount of biotin-probe made it difficult for the cold probe to kill the biotin-probe completely, I will try to perform supershift EMSA next to confirm this result further.

Thanks again for your nice and critical suggestions!

Sincerely,

Kuangxin