Yeast protein extract preparation for a quick western blot - (Aug/31/2007 )
I want to run yeast proteins on a gel just to see whether my protein of interest is expressed (qualitative WB). Can somebody give me a quick (can be dirty) protocol for yeast protein isolation? The ones I have from Clontech Yeast Handbook seem a bit too lengthy for my purpose.
My protocol is like this:
- Culture yeast o/n in proper medium (1-2ml)
- take 1ml, spin down, remove the supernatant
- Resuspend the pellet in 1ml of ice-cold H2O
- Add 150ul of Yeast Extract Buffer (YEX: 1.85M NaOH, 7.5% beta-mercaptoethanol); mix thoroughly and leave on ice for 10min. This YEX buffer I usually made fresh, otherwise keep at 4oC, dark.
- Add 150ul of 50% TCA, mix and leave on ice 10min. You can tap the tube from time to time.
- Spin at 4oC, 5min
- Resuspend pellet in 100ul of 2xSDS SB. The sample will turn yellow and cloudy.
- Add in 1-2 drops (about 10ul) Tris, pH 8 so that the sample turn back to the blue colour of SDS SB (SDS sample buffer), this is just to adjust the pH.
- Boil 3-5 min, 95oC
- Spin down and run gel. There will be precipitate in your sample. Don't worry, just run the sup is fine usually.
Hope it helps
Thanks a lot, I will try it! Just one question - do you run the whole volume of sup (~100 ul) in one lane of a minigel? I am just worried about overloading the gel. Appreciate if you could let me know.
No, I only run about 20-30ul. If too much, then reduce.
I ran a western using your protocol, it worked great! I can see my protein expressed and nothing in control cells. Thanks again!!!
You are welcome