Protocol for extraction of total protein from organs............ to do westerns .....!
alion85 at hotmail.com
Mon Jan 26 06:33:16 EST 2004
"exce-lent" <acb at plouto.net> wrote in message news:<AO_Qb.14552$zy3.8574 at okepread01>...
> I will appreciate of I could get a protocol for extracting total protein
> from tissues like liver, brain, kidneys, prostate, ovary etc, for doing
> western blot analysis.
> Any additional tips will also be appreciated.
No problems, you just have to homogenize your sample thouroughly, for
example with Dounce homogenizer (depending on your will to remain
nuclei intact or not use type B or type A pestle)... Then you could
review Dignam et al article dating back to 1983 in Nucleic Acids
Research... very effective protocol for doing extracts both
cytoplasmic and nuclear....but they are for NATIVE proteins..
generally Hepes_NaOH based ph7.5 buffers containing glycerol 25% and
some 420mM NaCl, DTT 0.5, PMSF 0.5 mM, MgCl2 1.5mM (this is just for
nuclei to remain intact)... etc .. you can put some protease
inhibitors also... then homogenize the tissue and sonicate some 10-15
pulses at output setting 4 for example 2 seconds pulse (actually this
is optional, Dounce will do its business), but the main thing is to
not give your solution too much heat, if you want intact proteins...
then centrifuge divide all the stuff as follows:
supernatant will contain native proteins (native extract) and the
pellet could be resuspended in Laemmli PAGE loading buffer (with 1/10
DTT [1M]), so you get the denaturated portion of the lysate with
PS. NaOH should be used to titrate your HEPES, :) i had a lot of
problems recently, titrated it with KOH, but those guys, i mean
POTASSIUM and Dodecyl Sulfate (Part of SDS) precipitate, and believe
me you dont want that)...
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