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western blotting question


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3 replies to this topic

#1 ismailhamad

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Posted 30 October 2004 - 12:45 PM

I can not perform SDS-PAGE and western in the same day so
1. Can I cast the gel one day before? (if it possible how can I preserve it?).
2. Or after SDS-PAGE can I perform blotting next day? (it possible how can I preserve the nitrocellulose fitler?)

#2 Simonsays

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Posted 01 November 2004 - 06:04 AM

You can cast your gel the day before and let it stand in the fridge overnight in runnig buffer. After the SDS-PAGE you can transfer overnight, or block your membrane overnight, or incubate it in the primary Ab overnight, or incubate it in the secondary Ab overnight... that's the beauty of Western...

Simon

#3 Sprag

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Posted 01 November 2004 - 02:31 PM

a) Yes, you can cast the resolving gel and leave in the fridge for days as long as you keep water on top so the gel doesn't evaporate. But for best resolution, cast the stacking gel fresh to preserve the shift in pH between stacking and resolving gel. That's why precast gels have an expiring date

:lol: You can easily keep your nitrocellulose membrane in blocking solution at 4 degrees over night, or block for 1h and leave in primary overnight shaking in the coldroom...

good luck,

#4 Oleksii

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Posted 03 November 2004 - 10:25 AM

You can even dry your membrane after transfer and store it at +4 up to one month. It is according manufacture instruction and it works!
:unsure:

Alexei




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