Western Blot problems

Daniel Harkness harkness at bio.bu.edu
Fri Jan 3 10:17:49 EST 1997


Laurence Pelletier (pelletl at IRCM.UMontreal.CA) wrote:
: I am currently having problems western blotting total yeast extracts i
: prpare using glass beads. 

: Here's the problem: Most of the time (9 out of 10) the blots are full of
: streaks

If one is making extracts, definately use a cocktail of protease inhibitors.
A potential reason for your 'streaks' could be degradation.  When purifying
GST-fusions from bacteria I always make a lysate that contains a battery of
inhibitors.  Also, if you don't need the extract for immunoprecipitation
(or similar protocol) then just bust open your cells in SDS sample buffer.
That will denature both your proteins and the proteases.  If you want, just
email me and I can give you the recipe for the protease-inhibitor cocktail.
I hope this helps...

Sincerely,
Dan Harkness
harkness at bio.bu.edu



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