5 replies to this topic

[intekmdma]

Hi all,

First time i'm posting (but have been a long time lurker)


I had to incubate my membrane at secondary antibody overnight at 4c because I found that we ran out of our detection reagents (for HRP).  I was wondering how many washes I should do to minimize background noise.

It's a cell extract so I'm guessing I may need more washes?

p.s. 1:10,000 2ndary if that helps.

Thanks in advance.

[Rute]

Well, I have done previously secondary ON at 4 degrees and did 5 washes of 5 min each... It did not show any background so I guess it workes that way..
However, I must point out that my primary antibody does not bind much. But I would stick to the 5*5.

Hope it helps

[intekmdma]

Thanks for the quick reply Rute.  I'll try that and update how it came out.

[Curtis]

why secondary overnight? one hour at RT is enough

[casandra]

Curtis, on 15 February 2012 - 11:26 AM, said:

why secondary overnight? one hour at RT is enough

because they ran out of ECL substrate (?)....

@ intekmdma: you can try incubating for one hour with the secondary and then keeping the blots in buffer O/N at 4 degrees... or incubate with the primary for one or two hours and then keep the blots in buffer O/N at 4 degrees then react with the secondary the following day... of course it depends on the antibodies, but if you really prefer to do an overnight incubation, it's probably better to incubate with the primary rather than the secondary....

Edited by casandra, 15 February 2012 - 08:38 PM.

[intekmdma]

casandra, on 15 February 2012 - 08:31 PM, said:

Curtis, on 15 February 2012 - 11:26 AM, said:

why secondary overnight? one hour at RT is enough

because they ran out of ECL substrate (?)....

@ intekmdma: you can try incubating for one hour with the secondary and then keeping the blots in buffer O/N at 4 degrees... or incubate with the primary for one or two hours and then keep the blots in buffer O/N at 4 degrees then react with the secondary the following day... of course it depends on the antibodies, but if you really prefer to do an overnight incubation, it's probably better to incubate with the primary rather than the secondary....

Hi Casandra,

Thank you for the wise words about keeping the blots O/N in buffer.  I have not received the ECL kit yet, and am expecting it tomorrow.  Not sure if the membrane which was sitting in secondary for 2 days at 4c is still good, so I went ahead and ran another gel letting it transfer O/N.

BTW: I didn't intend to incubate with secondary, I was just a bit too slow to grab the last of the detection reagents.