Western Blot Protocol

Western Blot Protocol (Standard, using Bio-RAD system)
Protocol is similar to Cell-Signaling Tech protocol

Notes: Either BSA or non-fat dry milk can be used to block blots. Primary antibody concentrations that are too high may not be completely washed away in first three washes and could bind secondary antibody and be subsequently washed away, lowering signal. Directly labeled HRP or biotinylated antibodies work best (with SA:HRP secondaries) and give lower backgrounds than anti-IgG secondaries, which usually give higher backgrounds. Two quick washes before the three 5 minute washes can be used to lower membrane background after secondary or primary if desired. Super high membrane background (membrane outline background) can be eliminated by washing membrane in wash buffer containing a small amount of sodium azide (0.03% final) for initial 1 or two washes after secondary incubation. Be sure to not use sodium azide wash buffer in final wash, it will greatly inhibit HRP activity in the presence of substrate.

Buffers/Reagents
•   Transfer Buffer: 25mM Tris Base, 0.2M glycine, 20% Methanol (pH 8.5)
•   10x TBS
•   Blocking buffer: 1X TBS, 0.1% Tween-20, 5% non-fat dry milk or BSA
•   Wash Buffer: 1X TBS, 0.1% Tween-20
•   Antibody Incubation Buffer: 1X TBS, 0.1% Tween-20, 1-5% non-fat dry milk or BSA (user determined for background vs signal)
•   ECL reagents (If using HRP), see protocols for homemade ECL

1.   After running gel, equilibrate all transfer materials in transfer buffer for 15-30 minutes.
2.   Assemble transfer apparatus. For Bio-RAD wet transfer, assemble in following order: clear side, sponge, filter paper, membrane, gel, filter paper, sponge, black side. Be sure to eliminate all bubbles and keep sandwich wet while assembling.
3.   Put sandwich into wet-transfer apparatus by matching black side of sandwich to black side inside transfer box and transfer at 100V for anywhere between 30 minutes and two hours depending on protein size and type of membrane used. Be sure to use ice-cold transfer buffer and an ice pack since transfer creates lots of heat. A stir bar in the bottom helps prevent hot zones.
4.   After transfer, wash membrane in 1X TBS at RT for 5 minutes.
5.   Incubate membrane in blocking buffer for 30 minutes to 1 hour at RT.
6.   You can wash the membrane after blocking or simply dump out blocking solution and add primary antibody solution. Concentration of primary should be determined for each antibody that is used.
7.   Incubate overnight at 4 C with gentle agitation.
8.   Wash membrane 3 times with Wash buffer, for 5 minutes each wash.
9.   Incubate membrane with secondary antibody (if appropriate), 1 hour RT.
10.   Wash 3 times with Wash buffer, 5 minutes each wash.
11.   Incubate membrane with ECL reagents of choice.
12.   Expose x-ray film with membrane, times vary greatly from 1s to overnight depending on strength of signal. Try to get multiple exposure times.
13.   Label film.
14.   Membrane can be stripped, re-probed or stored at 4 C.