Download Band Shift Crude cell extract

Yeast crude cell extract:
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Spin down ~30ml of culture of an OD600 ~0.4-0.5, wash with iced H2O,
remove all liquid and freeze on dry ice (or continue to step 3)
Thaw cells on ice for 10-15 minutes (no more)
Add 2ml/gram of cells of binding buffer + DTT & protease inhibitors to tubes
Vortex to resuspend pellet and add ~ ½ the volume of glass beads (425-600um)
The following step require a cold room and all tubes need to be pre-chilled:
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Fast prep machine: 4.5 speed for 30 seconds
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Transfer to ice for 3 minutes
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Repeat above steps a total of 5 runs
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Poke a hole into the bottom of the tube of lysed cells and spin transfer
the liquid into a 2ml tube
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Transfer liquid to a fresh 1.5ml tube without disturbing the pellet
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Spin 1.5ml tube for 30 minutes at ~14k RPM
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Transfer supernatant to a fresh tube = Protein Lysate
Check protein concentration and freeze in liquid nitrogen, store at -70˚C
1x Binding Buffer:
100mM Tris pH 7.5
100mM NaCl
0.5mM EDTA
2mM MgCl2
20% Glycerol
Protease Inhibitors:
1mM PMSF
1ug/ml Lenpeptin
1ug/ml Aprotpeptin
1mM DTT
25ug/ul Poly DIDC
Band Shift with crude cell extract:
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Label 10pmols of double strained oligos following the protocols in the Molecular
Cloning book
Pour a 17x20cm plate, 1mm thick, 5% 37.5:1 Acrylamide:bisacrylamide gel 1x TBE
o Pre-run the gel at 160 volts for ~1 hour (but no more)
o Binding mix:
 1x Binding Buffer
 10k-20k cpm/lane probe
 40ug crude cell extract
o Gentle mix and incubate at room temp for 30 minutes
o Add loading buffer and gentle mix
Wash lanes and load full reaction to gel and run 160 volts for 3-3 ½ hours
Transfer to blot paper and dry in a gel dryer for ~30 minutes
Expose to film for 2-4 hours at -70˚C