Western Blot Procedure and Discussion

[adapted from various sources]

The concise printable version is in the recipes section

Buffers:

Blocking Buffer PBSSM

1% non-fat dried milk
0.05 M phosphate
0.64 M NaCl
pH 7.4
0.1% TWEEN-20

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 (local grocery store's brand works well and is cheap); also other blocking agents are commonly used:

TWEEN blocker: just add another 0.5 ml TWEEN 20 to the 50 ml PBSST.

BSA: add 1.5 g bovine serum albumin to 50 ml PBSST.

Washing Buffer:

Use the left over 950 ml PBSST from above to wash.

Primary Antibody Solution:

Add your antibody solution to PBSST at your favorite dilution. Don't know what dilution to use? 1:1,000 seems to give a high probability of success for most antibody stocks. If you know the conc. Of antibody, try 1 ug / ml final dilution. Otherwise, the best method is to try a number of dilutions 1:200, 1:1,000, 1:10,000.

Secondary Antibody Solution:

Similar to the primary antibody, dilute in PBSST to the concentration given by the manufacturer (Usually between 1:1,000 and 1:20,000)

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.

Tips, Tricks and Hints

Temperature: Ambient temp. is usually fine for all steps; we tend to use four degrees for overnight antibody incubations

Experiment with the washing conditions! Some antibodies are messy and require stringent washes; others are easy to handle and the wash times can be easily reduced. If you are going to do a blot over and over again experiment with all times, primary, secondary, washing etc... You may be able to optimize your protocol to finish it in 2 hours from start to finish.

Make sure your secondary recognizes your primary antibody! Are you probing IgM instead of the usual IgG? Not all polyclonals are rabbit and not all monoclonals are mouse!

To speed up your blocking, PIERCE sells some excellent pre-made blocking solutions which effectively block within 30 minutes. (e.g. SuperBlock Cat #37515).

How does it work?

Initially, your blot has proteins spread onto it with ionic sites, and hydrophobic patches in bands. The first rinse with PBSST removes chemicals used in blotting and reduces spots, lines, and graininess. The blocking buffer then coats ionic patches with salt counterions and hydrophobic patches with either milk proteins, detergent, or BSA depending on the blocker used. The next wash removes excess milk proteins. The primary antibody solution is then used to allow your antibody to recognize and bind its target protein. The excess antibody is washed off, and a secondary antibody is added which will recognize the primary antibody. This step amplifies the signal and usually this antibody has an enzyme linked to it used for detection. Horseradish peroxidase (HRP) or alkaline phosphatase (AP) are the most common read-out enzymes covalently linked to the secondary antibody. These enzymes are then incubated in a substrate which gives either a colorometeric or a chemiluminescent signal which can be read by eye or by film.