disappear protein band after development - (Dec/19/2007 )

Dear all,

Hi all. I have an interesting problem about western blotting of my interested protein. I bought a new antibody and tried western blotting once. The target bands are quite obvious. However, after I repeat the experiment again with the re-used antibody, I can't get the bands again but some non-specific bands only. So, I incubated the membrane again with freshly prepared antibody the next time. Still no bands (target ones) there. No matter I prepared new samples or new antibody (not re-used one), the target band did not appear again....... ><

Do you know what's the possibilities of that? I have already aliquot the stock into several tubes. So, I think that is not a problem of freeze and thaw.

Thanks for all.
SamSam.

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Hi friend

To use again the membrane for WB, U PROBABLY did membrane striping, that might cause problem to ur membrane.

did u test ur striping protocol, in the past.

another possibility is problem with ur secondary Ab or Ab substare.

I think u should repeat the experiment with new samples and membrane

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look at what you did the first time and what you did after. are there any differences?

stocks? polymerization time? apparatus? power supply? membrane (lot#)? incubation time and/or temperature? anything?

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Hi Amtash,

Thanks for your information first. I also did repeat the whole experiment already. I mean new samples, new membrane and also fresh primary and secondary antibodies as well. But still can't get what I want just like the 1st time trial. So, this really makes me don't know the reason.

Sincerely,
SamSam

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Hi Mdfenko,

The difference is only the samples (old and new ones). But I have tried the sample that I used in my 1st time again, but still no target band on the membrane....

I used the same pack of membrane for transfer. and the incubation time and temperature are all the same in 4C with shaking as well. whatelse......nearly all the things are the same

Sincerely,
SamSam

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have you confirmed the transfer (stained the gel after transfer and stained the membrane with ponceau s)?

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Hi Mdfenko ,

I have stained the membrane with ponceau S already and find many bands there including my target size. And moreover, after development, the non specific are there but not my target one.

Sincerely
SamSam

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did you prepare a big stock of blocking solution? maybe it's contaminated and pH decreased.
you should always use fresh blocking solution, or add Na azide, but be careful with HRP-conjugated antibodies, it will kill the enzyme.

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i should have asked this before: did you see the nonspecific bands staining on the first western?

can you try stripping and restaining the first blot?

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Hi Missele,

For the blocking solution, I prepare it every time before blocking. So, I don't think it is the problem of the blocking solution.

Sam Sam

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