Western Blotting

Make RIPA buffer (to be used in 3 months after adding Nuclease)

A	B
50mM Tris HCl, PH 7.4	5mL (1M stock)
150mM NaCl	5mL (3M stock)
1% Triton X-100 or NP-40	1 mL
0.1% SDS	1mL (10% supply center solution)
1mM EDTA	0.2 mL (0.5M stock)
10mM Naf	0.042g (@L5P1 chemical room)
0.5% Sodium deoxylcholate	0.5g (@L5P1 chemical room)
	Add ddH2O to 100 mL
	Add PMSF [final] = 1mM and any other protease inhibitors immediately before use
	Add 1uL Pierce™ Universal Nuclease per 1mL

SDS Running Buffer

Trypsinize cells

Wash pellet with PBS

Remove all supernatant with micropipette

Freeze pellet in liquid N2

Thaw quickly at 37°C

Resuspend cell pellet on buffer 100µL

Vortex 0h 0m 15s

Incubate on Ice 0h 20m 0s

Vortex 0h 0m 15s

Collect supernatant

Centrifuge 10000 RPM for 0h 5m 0s

Collect supernatant without disturbing the pellet

Quantify protein

799 uL water + 1 uL lysate

• 200 uL 5x Bradford reagent

Read at 595 nm

Load 50 µg of protein/well

Loading buffer 50µL β-mercaptoethanol + 950µL 2x Laemli Buffer

Water up to 15µL

Boil at95°C before loading  0h 0m 15s

Add 10µLof ladder (Precision Plus Protein - Dual Color Standards)

Run at 120-150 volts 1h 0m 0s

Activate PVDF membrane in methanol

Soak filter paper in Towbin Buffer

Equilibrate gel in Towbin buffer 0h 10m 0s

Semi-dry transfer

90' (<50KDa) or 120' (>50KDa)

Mount sandwich (paper-membrane-gel-paper)

45 mA/gel (constant current)

Cut out LEFT UPPER corner of the membrane

Block membrane with 5% BSA in PBS 0.1% Tween (PBST) for 0h 30m 0s

Add primary antibody in BSA-PBST and incubate overnight 18h 0m 0s at 4°C

Wash 3x with PBST for 0h 5m 0s

Add HRP secondary antibody in BSA-PBST and incubate for1h 0m 0sat room temperature on a shaker

Wash 3x with PBST for0h 5m 0s

Add 500 µL ECL (enhanced chemilumescent) per membrane strip