Western Blot procedure
For additional information, read: Protein
blotting: a review, Biji T. Kurien aA biji-kurien@omrf.ouhsc.edu
and R. Hal Scofield a,b,c Journal
of Immunological Methods 2003, 274:1-15
Protocol
1) Cut 6 pieces of 3MM Whatman papers and 1 nitrocellulose membrane (to
the exact size of the gel).
2) Put the papers, membrane and gel in 1x Transfer buffer.
3) Arrange 3 papers in the transfer apparatus, the membrane, the gel,
and the another 3 papers on top.
4) Transfer 30-60 min 15V.
5) Colour the membrane with Ponceau S and wash with TBST.
6) Block 1h with superblotto (or ON at 40C).
7) Put 1st Ab into Ab incubation buffer. (For
anti-His 1:500-1000) Incubate 2hr RT or ON at 4oC. (1st
Ab can be reused, keep at 4 deg.with sodium-Azide).
8) Wash x3 with TBST (30 min).
9) 2nd Ab into Ab incubation buffer (1:5000 for
mouse HRP). 1h RT.
10) Wash x3 with TBST (30 min).
11) Develop with ECL: 2 ml 1xECL (buffer A)
2 ml 1xECL (buffer B)
12) Shake 1 min. Dry and develop.
1x Transfer buffer
100 ml 5x Running buffer
300 ml H2O
100 ml MetOH
Superblotto (200 ml)
5xPBS
40 ml
20% Tween 5 ml
Glucose
36 gr
50% glycerol 40 ml
BSA
6 gr
Milk (powder) 2 gr
H2O up to 200 ml
TBST (1 liter)
10 mM Tris pH 8.0
150 mM Nacl
0.01% Tween -20
Ab incubation buffer
5% milk (powder)
0.05% Tween-20
in PBS