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Incubating Western in primary for longer than 24 hours at 4C?


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#1 noodle

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Posted 01 June 2011 - 11:41 AM

Is it acceptable to incubate membranes in your primary antibody dilution (5% Milk, 1x PBS, 0.1% Tween) for 48 hours at 4C, rather than the usual 24h? I wanted to decrease the amount of antibody used at a given time and I was curious if providing a longer incubation period would work.

Thank you!

#2 casandra

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Posted 01 June 2011 - 12:43 PM

Is it acceptable to incubate membranes in your primary antibody dilution (5% Milk, 1x PBS, 0.1% Tween) for 48 hours at 4C, rather than the usual 24h? I wanted to decrease the amount of antibody used at a given time and I was curious if providing a longer incubation period would work.

Thank you!

well, you can always try it but don't be surprised if you will also get a lot of background....even the 24h O/N incubation can be too long for some antibodies....if you want to economise, you can either re-use them (add azide or filter) or reduce the volume you'd incubate your membranes with eg use sealable plastic bags cut to the exact dimensions of the blots and then add the minimum volume of the Ab solution- just enough to make full contact with the membrane...just flip them after an interval of time during the incubation....

Edited by casandra, 01 June 2011 - 12:53 PM.

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#3 noodle

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Posted 02 June 2011 - 06:46 AM


Is it acceptable to incubate membranes in your primary antibody dilution (5% Milk, 1x PBS, 0.1% Tween) for 48 hours at 4C, rather than the usual 24h? I wanted to decrease the amount of antibody used at a given time and I was curious if providing a longer incubation period would work.

Thank you!

well, you can always try it but don't be surprised if you will also get a lot of background....even the 24h O/N incubation can be too long for some antibodies....if you want to economise, you can either re-use them (add azide or filter) or reduce the volume you'd incubate your membranes with eg use sealable plastic bags cut to the exact dimensions of the blots and then add the minimum volume of the Ab solution- just enough to make full contact with the membrane...just flip them after an interval of time during the incubation....



Would reducing the typical amount of antibody used, (going from 1:500 to 1:1000 help to eliminate background for an extended incubation time?

#4 casandra

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Posted 02 June 2011 - 09:56 AM



Is it acceptable to incubate membranes in your primary antibody dilution (5% Milk, 1x PBS, 0.1% Tween) for 48 hours at 4C, rather than the usual 24h? I wanted to decrease the amount of antibody used at a given time and I was curious if providing a longer incubation period would work.

Thank you!

well, you can always try it but don't be surprised if you will also get a lot of background....even the 24h O/N incubation can be too long for some antibodies....if you want to economise, you can either re-use them (add azide or filter) or reduce the volume you'd incubate your membranes with eg use sealable plastic bags cut to the exact dimensions of the blots and then add the minimum volume of the Ab solution- just enough to make full contact with the membrane...just flip them after an interval of time during the incubation....



Would reducing the typical amount of antibody used, (going from 1:500 to 1:1000 help to eliminate background for an extended incubation time?

really good commercial antibodies for westerns you typically dilute more than 1:500 (perhaps for immunocytochemistry you do) and 1 to 2 hours of RT incubation is more than sufficient. Is this antibody so precious then (a gift from another lab, extremely pricey etc)that you want to stretch it out or it doesn't bind so well that you need an O/N incubation? If you really really want to find out, then just incubate a tiny strip of your membrane with the dilution you want...had you done this 2 days ago (instead of thinking and mulling about it and posting your query here), you would have been showing us the results by now? ;)
"Oh what a beauteousness!"
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