Western Blot

RIPA buffer (1ml)
Basic RIPA 940ul
25x Protease inhibitor 40ul
100x PMSF 10ul
20x NaF 5ul
20x Na3VO4 5ul

Running buffer
25mM Tris-HCl pH8.3
192mM glycine0.1% SDS (w/v)

Transfer buffer (4C)
12mM Tris-HCl, pH8.3
96mM glycine20% methanol (v/v)

Transfer buffer (2L)
Tris 11.6g
Glycine 5.8g
Methanol 400ml

TBST (2ml)
Tris pH7.6 40ml
5M NaCl 54.8ml
Tween 20 2ml

10% gel (15ml for 2 gels)
H2O 5.9
30% acrylamide mix 5
1.5M Tris pH8.8 3.8
10% SDS 0.15
10% APS 0.15
TEMED 0.006

Stacking (4ml for 2 gels)
H2O 2.7
30% acrylamide mix 0.67
1.0M Tris pH6.8 0.5
10% SDS 0.04
10% APS 0.04
TEMED 0.004

Harvest cells
Wash cell pellets with cold PBS
Transfer to eppie with 1ml cold PBS
Spin 2.6k 2min 4C
Add 1x lysis buffer into pellet (30-50ul, 5x cell vol). Mix and resuspend
Incubate lysate on ice for 30min, sonicate at output ~2-3W, 5s x3, vortex 10s every 5min
Spin 14k 15min 4C
Collect supernatant and determine protein concentration using BSA as standard

Measure protein concentration (for 96 well)
Standard curve
50ug/ml BSA(ul) 0 50 100 150 200
dH2O (ul) 200 150 100 50 0
Bradford assay reagent 50 50 50 50 50
Final amount (ug/250ul) 0 2.5 5 7.5 10

Sample prepare
Protein 1ul
dH2O 200ul
Bradford assay reagent 50ul

Resuspend 20-40ug protein in 3x SDS sample buffer
Adjust sample volume with dH2O (30ul for 10wells, 18ul for 15wells)
Incubate samples on 99C for 5min
Chill on ice 1min
Load gel

Run gel
Run stacking at 70V ~30min
Run resolving at 110V ~45min

While gel is running, soak all filter papers and transfer membrane in transfer buffer
After gel is done running, equilibrate gel in transfer buffer for 5-15min
Assemble transfer cassette, make sure no bubbles are trapped
Run transfer at 23V 60min
Stain gel with amide blue 30s, check for even transfer
Destain with destain buffer 5min
Wash with washing buffer 3x 5min
Place membrane in blocking solution for 1h
Incubate 5ml primary antibody on rocker
Wash 3x 5min
Incubate 5ml secondary antibody (mouse 1:2000, rabbit 1:4000)
Wash 3x 5min
Add substrate (ECL Plus), 2ml/ membrane
React in dark 5min
Remove membrane and drain excess liquid
Put in cassette and expose

To strip membrane
Wet membrane in washing buffer
Use enough stripping buffer to cover membrane
Shake (5-15min)
Wash 3x 5min