Western blot

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

(if in doubt, use a membrane on both sides of the gel, and select the one after transferring that has colored ladder on it)
 * 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) 

*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