Here you can find some of my protocols on :
For more protocols on yeast, see the "related articles" page; see also the links on yeast where i've made a selection of the best sites i use.
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Protein purification
Proteins are producted in E.coli and expression is under Lac operon regulation.
Cells are resuspended in 25mM TrisHCl pH 8.5, NaCl 150mM and 5mM beta mercaptoethanol (15 ml per 1.5 liters of 2TY liquid medium). Freeze-thaw 3 times in liquid nitrogen. (add proteases inhibitor) Sonicate 2 minutes in ice; 10 sec on, 7 sec off. Centrifuge at full speed (70000g) for 30 minutes. Filter the supernatent on 0.22um filters. Deposit the filtered supernatent on a NiNTA column. Wash untill OD280=0 with 25mM TrisHCl pH8.5, NaCL 150mM, beta mercapto 5mM, Imidazole 10mM. Wash untill OD280=0 with 10% of buffer 25mM TrisHCl pH8.5, NaCL 150mM, beta mercapto 5mM, Imidazole 300mM. Eluate with 25mM TrisHCl pH8.5, NaCL 150mM, beta mercapto 5mM, Imidazole 300mM. At this step, one can pass the protein on a ion exchange column or pass directly to the next step (for ion exchange column, dilute 3 times sample in TrisHCl 25mM pH 8.5). Visualize each step on SDS-PAGE. Concentrate the sample to pass it on a gel filtration column. I use a superdex 200 hiload (5ml of sample). Filter the sample on 0.22um before deposit it on the column. buffer 25mM Hepes pH8.0, NaCL 100mM, beta mercapto 5mM. Concentrate the protein for proper use.
yeast growth conditions
growth medium: (liquid) YPG at 28°C
Yeast extract (difco) 0.5% (5g/l)
Bacto peptone (difco) 0.5% (5g/l)
Glucose 3% (30 g/l)
yeast sporulation
Starvation of diploid yeast cells for nitrogen and carbon sources induces meiosis and spore formation. The sporulation process can be induced in cells growing either on solid or in liquid medium.
Sporulation in liquid media:
Sporulation Medium:
10 g/l potassium acetate
1 g/l yeast extract
0.5 g/l dextrose
yeast protein extraction
Preparation of mini-extracts from yeast cells. — Culture in 150 (50) ml of YPG medium ;Stop the culture at A600= 0.5-1 o.u./ml — Centrifugation 10000 rpm, 5 min (+4°C), remove supernatant; — Pellet + 30 (7) ml of ice-cold extraction buffer, vortex, transfer cell suspension in the 30ml tube; — Centrifugation 8000 rpm, 5 min (+4°C), remove supernatant; — Pellet + 7 (1) ml of ice-cold extraction buffer, vortex , (transfer 1ml of the cell suspension in the Eppendorff tube); — Centrifugation 8000 rpm, 5 min (+4°C), remove supernatant; — Pellet + 0.6 (0.3) ml of ice-cold extraction buffer, vortex; — Add 6 (3) ul of lOOmM DFP, vortex, keep tubes on ice; — Transfer yeast cell suspension in the Eppendorff tube (of 2ml for better cell breaking), containing 500 (200) ul of the glass beads been keeping on ice; — Vortex with maximal speed 10 times x 15 sec and keep tubes on the ice between every action; — Centrifugation 13000 rpm, 5 min (+4°C) and recover supernatant in the new tube. Keep on ice.
For B-galactosidase activity measurement dilute this extract 10 fold. For protein concentration measurements (Biuret) to take 10, 25, 50, 75, 100 ul of extract. — The rest of extract can be frozen in the liquid nitrogen. Extraction buffer: 100mM Tris HCl pH8, 10mM MgCl2, 1mM beta mercapto
Breakage with a "french press"; the cell disruptor machine at 2KBars. 1 mg of cells is resuspended in 1 ml of buffer (Tris 0.1M pH 8). centrifuge 15 min at 15000 g, 4°C. supernatant, about 20 mg/ml, is used for protein kinase assay. Fractionation of the extract can be done to reduce background.
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protein kinase assay
Kinase : 20 uM
MgCl2 : 2 mM
ATP : 500 uM, 3 uCi/ul
Buffer : Tris 0.1M pH 8
Yeast extract : 5 ul
total volume : 15 ul
Kinase is added last. The mix is incubated for 20 minutes at 30°C in a dry block. 3 ul of denaturing buffer
is added and the mix is boilled for 10 minutes at 95°C, then ran on SDS-PAGE until the small molecules are
washed away. The gel is plotted between watmann and saran and aotoradiographied over night. (dry is better).