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Reprobing with the same antibody

western blotting stripping secondary antibody reprobing nitrocellulose

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3 replies to this topic

#1 new2thelab

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Posted 09 June 2012 - 05:22 PM

Hi all. I'm a lab newbie and I need a little help. I completed three different Westerns and imaged all three but I found NO signal for any of my antibodies (three separate antibodies, incubated at room temp for 1hr; washings; secondary for an hour at room temp). I did a Ponceau stain and saw that the protein is there so I know that everything ran and transferred ok. I think I need to do my antibody incubation for a much longer time -- like overnight at 4 degree.

My question is: since I'm incubating with the exact same antibody, do I need to strip the blots and reblock and everything? Or can I just put the primary on and continue from there?

Thanks for the help!

#2 Kamran

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Posted 10 June 2012 - 02:58 AM

You need to reprobe your blots after proper stripping.

Moreover, if your primary antibodies have low affinities for epitopes then increasing incubation time may help getting some signal.

Best of Luck :)

#3 new2thelab

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Posted 10 June 2012 - 06:25 AM

So, I need to strip even though I will be reprobing with the SAME antibody?

#4 bob1

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Posted 10 June 2012 - 03:37 PM

No, you don't need to strip - in fact I never strip my membranes, it only reduces the signal you are likely to get by removing some of the proteins on the membrane.

Have you (or someone in the lab) used these antibodies before? if not, you may need to titrate the antibody to get the right concentration for your system.





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