3 replies to this topic

[cancergeek]

Hi Guys,

I let my pvdf sit in wash buffer for a little too long after I did my secondary antibody incubation and I have a feeling it might have reduced the signal for my protein of interest. Not sure if I washed too much of the 2ndary off or what. Would I need to strip and reprobe the membrane for my primary and secondary again? or would it be possible for me to just reincubate with my 2ndary and try to image again?

Thanks

[cellcounter]

cancergeek, on Jul 21 2009, 01:18 PM, said:

Hi Guys,

I let my pvdf sit in wash buffer for a little too long after I did my secondary antibody incubation and I have a feeling it might have reduced the signal for my protein of interest. Not sure if I washed too much of the 2ndary off or what. Would I need to strip and reprobe the membrane for my primary and secondary again? or would it be possible for me to just reincubate with my 2ndary and try to image again?

Thanks

There is no sure answer to this. Right thing to do would vary according to your antibodies and stringency of washes etc. Prolonged washing may have reduced the amount of bound primary antibody too. So, you decide whether you want to..

(1) strip and do both steps again,
(2) just start from primary or
(3) just start from secondary.

I would go ahead and develop the blot, and if it did not work, strip and do the entire procedure again.

[bob1]

I would do as Cellcounter suggested apart from, I wouldn't strip. I would just re-block, add more primary and secondary as you would normally and go from there.

[little mouse]

I would not strip (you will lose some antigens), if you think you have washed your antibodies, I would just re-do from the blocking step.