problem

Patrick F.H. Lai pfhlai at LOOKSMART.COM
Sun Jul 19 17:05:31 EST 1998


---- Begin Original Message ----
>When I am preparing samples for Western I do the following:

>Thaw pellet of gamma-radiation treated LCLs (~4,000,000 cells) at 37oC=
.
>Resuspend pellet in 200ul UTB buffer (9M urea, 150mM B-mercaptoethanol=
,
>50mM Tris pH7.5). Sonicate for 2x15 seconds, keeping cool, then centri=
fuge
>for 25mins at 35000 rpm at 10oC. 

>The problem is that after centrifugation I get this viscous bubbly lay=
er
>floating on the top of the centrifuged lysate. There is a pellet there=
fore
>it has been centrifuged properly. 

>Has anyone had this problem and do they know what the layer is? I don'=
t
>think that it is unbroken DNA/histone as it has been thoroughly sonica=
ted.
>The layer is quite viscous which suggests its protein but it is in 9M =
urea
>so it should have dissolved.

>If anyone can suggest what this is then can you email me at:
g.s.stewart at bham.ac.uk

>Thanks.
>G.Stewart.
---- End Original Message ----


Dear G.,

This is a wild guess, but what utensil did you use to hold your sample
when you sonicate ?
Is it some funny plastic ?

I used to sonicate my samples for my Westerns, too. (Way back when...)
I was using glass tubes at the time.
Once, I accidentally moved the probe of the sonicator too deep that it =

touched the glass.  Besides getting lots of foam, my sample turned 
 from milky white to dirty gray.  A post-doc in the lab suggested that
 I might have solubilized some of the glass.

I wonder if the bubbly top layer of your sonicated samples came from
the plastic of the test-tube you used.
Try sonicating without letting the probe of your sonicator touch the
tube.
Pls let me know if this helps...... I'll have some Westerns to do this =
coming winter.  :-)

Good Luck !






Hope this is useful info.   :-)


Patrick F.H. Lai  
Graduate Student, University of Toronto 
< PFHLai at looksmart.com >




      
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