Western (yeast) (read through 1st)
- 'Start 3ml ON in YPD of strains to western
- Dilute from ON into 25-50ml YPD (make all ODs the same starting, maybe about 0.2)
- Let grow 30C shaker until OD = ~ 0.5 - 0.8 (maybe around 3-6 hours, take aliquots for OD as needed)
- Spin down cells in large centrifuge in middle room (across from -80freezer) 1900rpm for 5min is ok
- pour off supernatant (ok if a little is left on, ~1ml), flash freeze in liquid N2
- store -80C until ready for next step
- Thaw cells on ice, transfer to 1.5ml tube, spin down, aspirate supernatant
- **Keep cells/prtn cold/on ice from here on out**
- add RIPA buffer + protease inhibitors (make using 10ml ripa and adding protease inhibitor tablet) OR USE CELL LYTIC Y (from Sigma and skip freeze thaw and beads)
- add small glass beads, votex ~20sec, put on ice, do this 2-3 times
- Flash freeze by dipping into liquid N2, thaw on ice, vortex 10sec, repeat 5X
- Spin down 10 min full speed in cold room
- remove supernatant to new tube (discard pelleted debris)
- Ok to freeze here -80C and store
- perform BCA assay
- Calculate the volume of each sample to use to load somewhere between 20-50ug (we did 50ug last time). Remove aliquot for loading, add 4X sample loading buffer with reducing agent added (you’ll have to make a small aliquot of load+reducing, they are in small 4C by PCR machine).
- Load ladder & samples onto protein gel and run at 200V until loading dye nears bottom (maybe ~1hr) use running buffer 1X (don't forget ladder)
- Setup transfer - 4 sponges, filter paper, gel, membrane, filter paper, 4 sponges (RED end)
(if in doubt, use a membrane on both sides of the gel, and select the one after transferring that has colored ladder on it)
*Activate PVDF membrane by putting in Methanol for 30sec before transfering
transfer at 100V for 1.5hr or overnight at 20V use 1X transfer buffer
- remove MB from transfer (wash transfer rig, sponges, etc w/ DI water):make sure ladder transfered to MB
- Block (5% milk in TBST) for 1 hour RT
- wash 3X TBST
- add primary antibody (e.g., HSP60) let incubate 4C ON on roller
- SAVE primary antibody, pour back into tube and store 4C
- wash 3X TBST
- add 5% milk in TBST and add 2ndary antibody ~2ul (goat(or whatever) anti-mouse for HSP60, actin)
- RT for 1 hour, shaking or rolling
- wash 3X TBST
- mix aliquot of developing reagents, let MB sit in dev solution for 1 min
- put into cassette in plastic, bring film, and develop on 3rd floor developer (maybe start with 1min exposure and adjust longer/shorter as necessary)
- strip and restart at primary step
abbreviations RT=room temp, ON=overnight, MB = membrane