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quick Western and HA Ab

Geraldine Butler gbutler at macollamh.ucd.ie
Wed Oct 18 04:07:15 EST 1995

In article <tomasd-0609560945320001 at fp5-9n.domain>,
tomasd at bdg10.niddk.nih.gov (Tomas Drgon) wrote:

> 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
> Hi.
> 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.
> Tomas Drgon
> -- 
> Signature under construction...

I'm interested in the reference to the Boehringer antiHA antibody, at a
dilution of 1:10000.  The instructions with this antibody say to use 5ug/ml
- as they supply 200ug in a standard pack, this doesn't allow many
westerns! Have many people tried it at dilutions such as 1:10000?
Geraldine Butler  University College Dublin   Ireland

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