Western blotting analysis

Reagents

Lysis Buffer

50mM Tris-HCL

1% NP-40

0.1% SDS

0.1% Deoxycholic Acid

0.1mM EDTA

0.1mM EGTA

*Per 10mL Lysis Buffer, add fresh

2.25mg NaF

6.3 Na pyrophosphate

10mg Complete

2.7mg Pefablock

50uL of 200mM Na3V04

Sample Loading Buffer (6x)

70mL 4x Tris-HCL / SDS Stacking Buffer pH 6.8

36mL Glycerol

10g SDS

6 ml 2-ME

40mg Bromophenol Blue

Add water to 120uL

SDS Running Buffer (10x) (0.2501M Tris Base, 1.924M Glycine, 0.03467M SDS)

Transfer Buffer (25mM Tris, 192mM glycine, pH 8.3, 20% MetOH)

1% Casein in TBS (Biorad, 1610782)

TBST (20mM Tris, 150mM NaCl, 0.1% Tween20)

TBS (20mM Tris, 150mM NaCl)

Protocol

Gels

1. Make gels according to following recipe

Lysis

1. Lyse cells or tissue with cell lysis buffer. For cells, scrape into Eppendorf tubes on ice.
2. Sonicate lysates x5 times with short 2sec bursts
3. Spin at 14,000 rpm for 10min at 4℃
4. Transfer supernatant to new Eppendorf tube and quantify protein
5. Dilute all samples to same concentration and add sample loading buffer to x1, depending on volume
6. Boil for 10min @ 95℃
7. Spin for 30sec at 14,000 rpm and store in -20℃ or run directly on gel

Western Blot

1. Specifically for SREBP2 blotting, load 50ug of protein into gel. For other protein targets, load 20ug
2. Run gel at 80V for 30min in x1 SDS running buffer until dye front reaches resolving and then run for another ~2hr, until the dye front has completely run off the bottom of the gel

Transfer

1. Transfer blots to nitrocellulose membranes using transfer apparatus. Run for 2hr at 100V in 4℃ cold room

Detection

1. Block membranes in 1% casein for 1hr @ RT
2. Incubate membranes with diluted antibodies (1:100 in 1% casein) for 16hr rocking @4℃
3. Next day, wash x3 for 5min with TBST @ RT
4. Incubate membranes with diluted secondary antibodies (1:10,000 in 1% casein) for 1hr @ RT
5. Wash x3 for 5min with TBST
6. Was x1 for 5min with TBS
7. Read on Licor Oddysey CLx