How to increase expression of protein in yeast? - Western from yeast and liquid beta-galactosidase assay (Jun/13/2008 )
This one is for Y2H experts. I am novice in the Y2H field.
I transformed the AH109 yeast with plasmid of my interest. I am checking the expression of my gene of interest. Here is the Western snap.
Lane 1: Protein Marker
Lane 2: Protein of interest cloned in pGBKT7 inframe with c-myc
Lane 3: Negative Control (AH109)
Lane 4: Positive Control (pGBKT7-53)
Primary Antibody: c-myc mouse monoclonal
Question: How can I increase the expression of protein of my interest in yeast. Or is there any way to show healty band of my protein? Waiting for your responses. Will be happy to supply more information.
Is there anybody currently doing "liquid beta-galactosidase assay" for quantifying protein interactions in yeast? Will be happy to have some protocols and cool tips.
What kind of protein extraction have you done?
Here my favorite protocol for checking protein expression of Y2H strains:
centrifuge 3 OD cells, wash once with (1 mL) water, aspirate supernatant. Add to the pellets 1 microspoon Hcl washed glassbeads and 100 µL preheated Thornerbuffer with 5% ß-Mercaptoethanol and Protease-inhibitors (Thorner-Buffer: 8M Urea, 5% SDS, 50 mM Tris-Hcl pH 6.8) vortex, shake 10 min @ 70°C, vortex, spin down for 10min @ 4°C and 13000 g. From the supernatant I mix 10 µL with 5 µL 3xstop and load it on a SDS Gel. This normally gives good protein bands.
For quantitative ß-Galactosidasa assay, I used the protocol from the clontech yeast handbook
This works quite fine (as long as you always monitor positive and negative controls also)
The different measurements values from different preparations somehow were not really comparable.
But if you calculate as percentage activity from the positive control (=100%) it was OK.
I hope I could help a bit