Crazy ChIP! Background probs and All the sudden no signal! - (Jun/28/2008 )
What the!?
This ChIP is driving me crazy.
I was having background problems (I thought) before, and tried everything to fix it. Come to find out my background was fairly normal, my ab samples just had weak signals (my PolyII controls were fairly strong). So I got some different abs, tried some different amounts of ab. All the sudden I get no signal at all!! My inputs have plenty of signal, so I know I had sonicated DNA.
I did nothing different this time? Has this happened to anyone? The only thing different I did was use a vacuum apparatus to aspirate between washes of the beads (instead of pippette tip - very laborious). The vacuum did suck of better (leaving only the beads). Could it have sucked off to good? What the!?
Any help here?
Without being really helpful, I can answer this one. EVERYSINGLEDAMNEDONEWHOHASEVERDONETWOCHIPS.
This ChIP is driving me crazy.
I was having background problems (I thought) before, and tried everything to fix it. Come to find out my background was fairly normal, my ab samples just had weak signals (my PolyII controls were fairly strong). So I got some different abs, tried some different amounts of ab. All the sudden I get no signal at all!! My inputs have plenty of signal, so I know I had sonicated DNA.
I did nothing different this time? Has this happened to anyone? The only thing different I did was use a vacuum apparatus to aspirate between washes of the beads (instead of pippette tip - very laborious). The vacuum did suck of better (leaving only the beads). Could it have sucked off to good? What the!?
Any help here?
I doubt your problem is due to the aspiration unless you're losing beads using the vacuum to aspirate. Is there any chance you switched to a new vial of antibody (from a different lot). We've had problems with some lots on polyclonals not working because the company had to switch to using a new animal (since the original one probably died). If you've made any new buffers you might want to re-check the pH also.
Completely agree with KPDE, I previously experienced getting a new vial of the same Ab but a different lot from Abcam, and suddenly getting no signal. Luckily, I had given an aliquot of the old lot to a labmate, so I got some of the old stuff and ran parallel with the new lot, and it was obvious from the results that the new lot of Abs I just bought were just duds, and just weren't capturing anything (might as well have used it like IgG... LOL). If you still have the old one, you can try that and see if it's the problem. most companies will refund you if that is the case, or if they still have vials from the same lot they will send you that.
Good luck!
This ChIP is driving me crazy.
I was having background problems (I thought) before, and tried everything to fix it. Come to find out my background was fairly normal, my ab samples just had weak signals (my PolyII controls were fairly strong). So I got some different abs, tried some different amounts of ab. All the sudden I get no signal at all!! My inputs have plenty of signal, so I know I had sonicated DNA.
I did nothing different this time? Has this happened to anyone? The only thing different I did was use a vacuum apparatus to aspirate between washes of the beads (instead of pippette tip - very laborious). The vacuum did suck of better (leaving only the beads). Could it have sucked off to good? What the!?
Any help here?
I doubt your problem is due to the aspiration unless you're losing beads using the vacuum to aspirate. Is there any chance you switched to a new vial of antibody (from a different lot). We've had problems with some lots on polyclonals not working because the company had to switch to using a new animal (since the original one probably died). If you've made any new buffers you might want to re-check the pH also.