Silver: Lysate for Western: Difference between revisions
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#Grow cells | #Grow cells | ||
#Harvest | #Harvest | ||
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#Spin down at maximum speed in tabletop centrifuge for 10 min. (in cold room) | #Spin down at maximum speed in tabletop centrifuge for 10 min. (in cold room) | ||
#Remove supernatant (lysate) to another tube. | #Remove supernatant (lysate) to another tube. | ||
[[Category:Protocol]] | |||
[[Category:In vitro]] | |||
[[Category:Yeast]] | |||
[[Category:Protein]] |
Latest revision as of 11:12, 9 May 2007
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- Grow cells
- Harvest
- Spin down
- Wash with PBS
- Spin down enough cells to collect a 50-100 µL cell pellet in a FastPrep tube
- Prepare lysis buffer (beforehand) and chill on ice
- PBSMT:
- 2.5 mM MgCl2
- 3 mM KCl
- 0.5% Triton X-100
- in PBS
- Add protease inhibitors
- PMSF: 1 mL per 100 mL buffer
- Use fresh PMSF: dissolve 0.035g per 1mL 100%EtOH
- PLAAC: 100 µL per 100 mL buffer
- 0.5 M Benzamidine: 260 µL per 100 mL buffer
- PMSF: 1 mL per 100 mL buffer
- PBSMT:
- Add 50-100 µL of cold lysis buffer to cell pellet and vortex
- Add 1-2 scoops glass beads (bead level should be just below liquid level)
- Mix everything together by vortex
- Lyse in FastPrep bead beater
- 6.5 m/s
- 40 second lysis
- Add 500 µL lysis buffer (keep everything on ice!)
- Spin down at maximum speed in tabletop centrifuge for 10 min. (in cold room)
- Remove supernatant (lysate) to another tube.