Protocol: Full Membrane Western Protocol

Description: A generalized Western Blot protocol for use with full membranes (8.3 cm x 7.3 cm).

Lab: Paulovich Lab, Fred Hutchinson Cancer Research Center

Author: Rgi

Date: 01 November 2007

Procedure:

1. Place Membrane in 50 ml Falcon tube, protein side toward center.

Note: Add all solutions to the bottom of the tube without contacting the membrane.

2. Block: 7.5 ml Blocking Buffer (see below) / 60 min. / Room Temp (RT) / Rotisserie.
3. Remove Blocking Buffer by aspiration- use aspiration to remove all solutions throughout the procedure.
4. Dilute 1^o Ab in Antibody Dilution Buffer (see below).
5. Incubate 1^o Ab: 7.5 ml diluted 1^o Ab / overnight / 4^oC / Rotisserie.
6. Wash: 2 x (10ml Wash Solution / 5 min. / Rotisserie).
7. Dilute 2^o Ab in Antibody Dilution Buffer.
8. Incubate 2^o Ab: 7.5 ml diluted 2^o Ab / 60 min. / RT / Rotisserie.
9. Wash: 2 x (7.5 ml Wash Solution / 5 min. / Rotisserie).
10. Substrate: 7.5 ml 1x LumiGLO + Peroxide substrate / 10 min. / RT / Rotisserie.
11. Expose to film.

Reagents:

SuperBlock®: Pierce, Cat# 37515

Tween 20: Sigma, P7949

10X PBS, pH 7.4: Fisher Cat.# BP399-4

20X LumiGLO® Reagent and 20X Peroxide, Cell Signaling Technology Cat. #7003

Equipment:

Membranes: 0.45 mm Nitrocellulose, Invitrogen Cat.# LC2001

50 ml Falcon Tubes: BD Falcon BlueMax 50mL Graduated Tubes (352070), Fisher Cat.# 14-432-22

Rotisserie: Barnstead/Thermolyne, Labquake Rotisserie Hybridization Rotator, Fisher Cat.# 13-687-69

Solutions:

Block Buffer: SuperBlock with 0.1% Tween 20

Wash Solution: 1x PBS, 0.1% Tween20

Antibody Dilution Buffer: 1x PBS, 10% SuperBlock, 0.1% Tween20

Substrate: 1 x Lumiglo in H₂O.