Monday: 15/03/21
Add 1M DTT in each tube to final concentration of DTT 50mM Heat samples @ 70°C for 10’
Spin @ top speed (~21k rcf) for 1’
Run 20-well 4-12% Bis-Tris gel using MOPS running buffer
Load 25ug of total protein of each sup fraction and load same vol. of corresponding pellet fraction (total protein conc for these samples: )
Wet transfer, 70V for 1.5hr
Block in TBST/5% Milk @ RT 1 hour or @ 4C overnight/for the weekend
After blocking, rinse membrane with TBST until water runs clear
Add antibody and incubate @ 4C overnight (for the western, you can use the antibody from the ammonium sulfate suspension at 1:1000)
Do Bradford Assay of total protein concentration on each sup fraction to determine WB loading amounts
Tumor samples = 16.8 mg/ml (average) = load 1.5 uL (for 25 ug)
Normal samples = 22.5 mg/ml (average) = load 1.1 uL (for 25 ug)
Sample/(Concentration-mg/ml):
T1/(13.6)
T2/(17.2)
T3/(17.1)
N1/(21.6)
N2/(22.7)
N3/(23.3)
Resuspend beads in 50% Glycerol/1xPBS/0.5mg/ml BSA for long-term storage at -20°C
Tuesday: 16/03/21
All samples to be combined with 50 mM DTT (1x), heated @ 70°C for 10’ with then cooled and spun before loading.
Remove and save primary antibody at 4C
Wash membrane 3x 10 minutes in TBST on orbital shaker
Change box at second wash step
Remove last drops of TBST with vacuum
Add secondary antibody and incubate 1hr RT on shaker
Discard secondary antibody
Wash membrane 3x 10 minutes in TBST on orbital shaker
Change box at second wash step
Remove last drops of TBST with vacuum
Add 1-2ml of Luminata Forte HRP developer
Incubate 1-2 min, tilt box to make sure membrane is completely covered
Develop membrane on ground floor Amersham imager