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lysate concentration


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#1 Miriamtosetto

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Posted 15 December 2004 - 03:15 AM

Hi all,
I'm trying to concentrate my lysate with acetone but I find hard to dissolve the pellet in lysis buffer. I need to apply BCA assay to know the concentration again before to run my samples in a gel. Does anyone know another method or can give me the protocol with acetone to compare. I know I can buy colums but I have too many samples and they are too expensive.
Thanks million
Miriam

#2 maga

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Posted 17 December 2004 - 06:15 AM

TCA precipitation in 1,5 ml tubes:

add ice cold 20% TCA to a final concentration of 10%
let stand on ice for at least 3 min
spin down at 13.000 rpm, 10 min, 4 C (alternatively, use 8.000 rpm, the yield is the same, but the pellet is less tight, which might help resolve it later)
carefully remove ALL TCA

add 800 l ice cold 80% acetone to the pellet, vortex thoroughly (sometimes, it let it vortex in the cold room for 30 min; I try to get the pellet off the tube)

spin down, add acetone, vortex, spin down again

let the pellet dry in an open tube at 56 C (10-20 min); if you can still smell the TCA, repeat the washing steps; if you smell acetone, let it dry longer.

Add sample buffer and boil (I use 95 C)
We use sample buffer with bromphenol blue. If the sample buffer (or just the TCA pellet) turns yellow, the pH is too low due to TCA, and you will NEVER get it dissolved. In this case, add a tiny amount of NaOH. The vapor might suffice. Be aware that this might interfere with running behaviour in the gel.




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