Berglund:Western Blot

MBNL Western

Cell Treatment
Ripa Buffer (final concentrations)
25mM Tris HCl pH 7.6
150mM NaCl
1% NP-40
1% Na deoxycholate
0.1% SDS
make in amber bottle, store at RT
****This is a 1.1x solution
fc= 1x after addition of inhibitors

0.1M PMSF
wear a mask when handling PMSF
use 100% (200 proof) EtOH, make about 5ml of solution
vortex to dissolve
****this is a 100x solution (sometimes viewed as 50x)
store at -20
stable for 1 month, may crash and have to be resuspended

SigmaFast protease inhibitor pill
Dissolve 1 pill into 10ml water
store in fridge, it is never fully suspended, shake before using
undissolved pills are good for 4 years
dissolved pills are good for at least 2 weeks (good for 1 month)
***This is a 10x solution

1) Check the plate for confluency
2) take to lab, and prep the buffer
3) 24 wells = 30 ml RIPA, 30µl of PMSF and 300µl of SigmaFast all on ice
4) aspirate the media, do not wash cells, add 100µl Ripa complete to each well
5) shake in cold for 10 minutes on speed 6
6) place in eppendorfs and can be stored at -20°C over weekend
7) or (and this has to be done before using to complete lysis) vortex in cold on speed 4 for 15 minutes
8) spin at 12,000rpm for 15 minutes in cold
9) transfer supernate to new tubes on ice and freeze at -20°C

Gel
1) Pour 10% SDS protein gels (15 well combs)
2) take 12µl of lysate and add 3µl of 5xSDS loading dye
3) heat for 3 minutes
4) spin down for .5 min
5) load onto gels 12µl
6) load 1µl of PAGE ruler prestained ladder
7) run until dye is almost off
8) rinse rig, open plates, cut off stacking gel

Western
1) find the black and clear plate western sandwich cassette rig
2) open like a clam and put into a dish of 10% methanol/1x transfer buffer
3) place in order on the black side:
sponge
2 pieces of whatman paper
gel on of whatman
(shiny side faces gel) nitrocellulose, 0.45µM (roll) no touch
2 pieces of whatman (roll)
sponge
clear side
clamp it all down
4) place in rig, black side to black side
5) add ice pack
6) fill with 10% methanol/1xtransfer buffer
7) run in cold room for 1 hour at 500milliAmps
8) dispose of buffer in container under sink
9) pull rig apart
10) rinse staining dishes with methanol, collect the flowthrough for proper disposal
11) do the Ponceau stain
12) replace stain into bottle and then take a picture of the gel
13) put membrane in box shiny side up, destain in TBST (throw down drain)
14) Block 1hr at RT with 5 ml or so of Odyssey blocking buffer you can reuse this for your 1°

Antibodies
primary are light stable
secondary are fluorescent and unstable, treat accordingly
Primary goes on for 1 hour to overnight
Secondary goes on for 1 hour
Wash post 1° and 2° with TBST: 1 min, 1min, 5 min, 10 min
always pour out of the same corner
handle only with special forcepts, never touch the membrane
label membranes so they don't get mixed up. Can be stored up to 1 yr.or longer!!

primary: 200µl 10% Tween 20
(2 gels) 10µl MBNL Millipore
7µl GAPDH
10ml Odyssey blocking buffer

secondary 200µl 10% Tween 20
(2 gels) 0.7µl Mouse 800
0.7µl Rabbit 700
10ml Odyssey blocking buffer