19
edits
Changes
Corrections to protocol
==Protocol==
#Turn on heat block to 85 degrees
#Run SDS-PAGE gel using [[ SDS-PAGE Running Buffer ]] and prepare diluted transfer buffer
## Use a prepared 54-12% tris gel (in the 4 degree). Remove it from the packaging, remove the white strip of tape from the bottom back, and gently pull the comb out and rinse with water.
##Load into gel tank. Fill with SDS page Running buffer (1X) to the fill line in the front and halfway up the back
##Boil sample at 85 degrees for ~3 min##Load 3 microliters of protein ladder (purple) (in the 4 degree), and 10 microliters of each sample into separate wells.
## Place top on tank, plug into power source and run at 125 Volts until samples and ladder reach the bottom of the gel
#Make sandwich (black side, sponge, filter paper, gel, nitrocellulose, filter paper, sponge, clear side), ensuring no bubbles between layers with black piece on bottom and layer as above. Place in apparatus so that the black sandwich touches the black transfer piece. Fill with transfer buffer.
#Transfer 4h at 75V (in cold room) or overnight at 35V (room temp).
#Stain for total protein with Revert total protein stain on rocker for 5 minutes --when finished pour total protein stain back in bottle for later use!
#Rinse twice in revert wash solution (60ml MeOH, 13.4 ml Aceditc Acetic Acid, 126.6 ml Water)
#Scan using licor for total protein, which will be used to normalize the blot
#Rinse nitrocellulose in revert reversal solution for at least 5 and no more than 10 minutes until nitrocellulose appears clear again (.2g NaOH, 60ml MeOH, 140ml Water)