In article <01HWDW80DR8K93C5JR at Gems.VCU.EDU>, ROSENKRANTZ at Gems.VCU.EDU wrote:
> Does anyone have a reference or protocol for a quick yeast Western and
> a recommended source of HA Ab? Thanks
Take yeast suspension in 0.05M Tris pH 7.4 + protease inhibitors, about
0.5 to 1 g wet weight per ml. Put in 10-15 ml glass testtube, add glass
beads in a way that there will still be about 4 mm of liquid above the
beads. Vortex full speed 5x30 secs/30 secs on ice. Spin down the beads +
cell wall debris + unbroken cells 5 min at 2000 g. Collect the sup. You
can further fractionate by differential centrifugation (I usually do a 15
min 40 000 spin in Beckman). Determine proteins, and apply 20 - 40 ug per
well on a usual minigel (Novex).
I use mouse antiHa from Boehringer (cat#1583 816), 1:10000 for 30 min at
RT, 2-nd antibody goat anti-mouse coupled w HRP, nickel enhanced
Diaminobenzidine staining (Red Book).
Let me know if you have any further questions.
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