Supershift - (Nov/02/2006 )
Can you help me?!!
I can't visulalized my supershift band with my GR antibody 1 µg. I add the antibody to the binding buffer with nuclear extract (5µg) 30 min at room temperture, and i add my probe 20min. I tested two different binding buffer with or without BSA but i have the same probleme.
I tested incubation of antibody with nuclear extract over night at 4°C but no result. Can you help me to resolve this problème. Thank you. 
Supershift is sometimes difficult. You can try to incubate for 1h at RT with your antibody before adding the probe. And you could try to add the probe first and then the antibody. I have also read about adding 0,05% NP-40 to the supershift reaction.
Good luck.
I would try more than 1ug. I know that seems like a lot, but if you are looking for a rare interaction then perhaps it would do to add more? I have added up to 5ug to get a stubborn supershift to show (just make sure to do a neg. control to overrule nonspecific binding)
Good luck.
Thank you, I will try to incubate 1H at RT. I have already tested to incubate brobe and antibody in the same time 20 min at RT but no result. To day I try differents concentration of antibody: 1µg,2µg and 4µg 20min at RT before adding the probe, I show you the result next Monday.
Thank you for your help.
Is agitation necessaire for supershift?
what do you mean, agitation?
I add all components to the bottom of the tube and then flick it twice, then tap it gently to make sure the contents are in the bottom just prior to incubation....do you normally vortex yours?? 
I add all components to the bottom of the tube and then flick it twice, then tap it gently to make sure the contents are in the bottom just prior to incubation....do you normally vortex yours??
thans for your reply, yes i vortex my mix also normally but gently. I tested recently 1h at RT of incubation with antibody 1µg, 2µg and 4µg with differents binding buffer with BSA or NP-40 1% + BSA but no result. I have just a decrease of bande protein/ADN but without the supershift bande!!
Because I can't visulalized my supershift band, Can I electroblotted my gel shift onto a membrane and identified my proteine GR/probe with antibody anti GR on the membrane after transfer?