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


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