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0.5 OD=500 ul cells=1 gel lane
10X IP Buffer -SDS
- Grow yeast cells to 0.5-1.0 OD.
- Pellet cells and resuspend in YM media (+a.a. but minus methionine) at 1.0 OD/ml.
- Put cells in 15 ml snap top tubes and grow for at least 15 min. with shaking.
- Add 35S label at 100 uCi per 0.5 OD.
- Pulse for desired amount of time (typically 10 min.).
- Chase label by adding 5 ul per 500 ul of cells of cys/met mix (5 mg/ml each).
- Harvest desired number of OD's at chase times in 2 ml tube.
- Add 100 ul SUME + protease inhibitors.
- Add equivalent amount of glass beads.
- Vortex 1 min. 3 times by hand and remove lysate to 1.5 ml tube.
- Clarify lysate with 5 min. spin and remove to new tube.
- Clarify lysate again and remove to new tube.
- Add 100 ul of 10X IP buffer -SDS, 800 ul H20, 1/10 ul antibody.
- Incubate overnight at 4°C on nutator.
- Add 110 ul of 10% Protein A-Sepharose to each sample.
- Incubate for at least 1 hr. at 4°.
- Spin 1000 rpm for 15 sec., remove lysate and save.
- Wash sepharose 3 times with 1 ml of 1 X IP buffer +SDS.
- After final spin, aspirate to dryness with blue (25G) needle.
- Resuspend in 30-50 ul 2X USB. Heat samples at 65°
C for 5 min. and load onto gel.
- After running gel, incubate in fix for 30 min.
- Rinse gels thoroughly and incubate in Amplify for 15-30 min.
- Incubate gel for 5-10 min. in 2% glycerol.
- Dry gel and expose to film.
0.9 M Tris-HCl pH 8.0
1% Triton X100
20 mM EDTA
1X IP Buffer +SDS
0.1% Triton X100
2 mM EDTA
10% acetic acid