Flaws in this protocol?

U. Gausmann ugau at mti-n.uni-jena.de
Thu Apr 30 11:56:20 EST 1998


Hi Zach,

I  used a quite similar protocol for SDS-PAGE of yeast extracts.
But I always included a neutralization step after TCA precipitation.
If your extracts turn yellow much of your protein will be degraded.

Neutralization is quite simple:
Put 100ul or so of a 1M Tris solution (pH NOT adjusted - it is around
11 I think) onto your pellet - immidiately after centrifugation and
quick removal of the acid. Do NOT resuspend.
Remove Tris and treat pellet as your protocol indicates (for me acetone
treatment was not necessary - I reuspended my protein directly in SDS
loading buffer).

Perform all steps on ice and cool your cells while treating with
NaOH/Mercapto.

Hope this will be useful,
Ulrike

--
Ulrike Gausmann           | D-07743 Jena
Institut fuer Anatomie    | Germany
Anatomie II               | Tel +49 (0)3641 938553
Teichgraben 7             | email ugau at mti-n.uni-jena.de





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