Western Blot [adapted from various sources by http://www.proteinchemist.com ]

Buffers:

Make one liter PBSST:

Add 12.19 g of dibasic sodium phosphate (Na2HPO47H2O), 0.62 g monobasic sodium phosphate (NaH2PO41H2O) and 37 g NaCl to 1 liter of water. Simpler, use 5 instant PBS tablets (Sigma, P4417), and 29 g NaCl in 1 liter water. Add 1 ml TWEEN-20, stir to dissolve. Use 50 ml of this to make blocking buffer by adding 0.5 g non-fat dried milk powder

Blocking Buffer: Add 0.5 ml TWEEN-20 or 0.5 g non-fat dried milk to 50 ml PBSST
Washing Buffer:
Use the left over 950 ml PBSST from above to wash.
Primary Antibody Solution: Dilute your antibody to the recommended dilution in PBSST
Secondary Antibody Solution: Dilute in PBSST to the concentration given by the manufacturer

 

Procedure:

Rinse your blot in wash buffer after blotting, add blocking buffer, incubate with gentle agitation for 45 min. to 2 hours.

Wash 1: Pour out the blocking buffer (can be reused), add washing buffer, agitate on a shaker for ten minutes. Remove washing buffer, repeat two more times. (Can be done with four steps of five minutes each)

Probe: Add primary antibody solution to blot. Incubate for 2 hours to overnight with gentle agitation. Pour off primary antibody solution (Can be reused).

Wash 2: Repeat wash as described in wash 1.

Secondary probe: Add secondary antibody solution. Incubate for 1/2 to 1.5 hours with gentle agitation. Pour off secondary antibody solution (Can be reused).

Wash 3: Repeat wash as described in wash 1.

Develop: Develop using the method described by the maker of your secondary antibody.