/

General Western Blot Protocol

Many variations exist among protocols for running Western blots. These variations address all aspects of the procedure from the choice of gel buffers and membranes to transfer conditions, blocking and processing steps. The following procedure was written as a general protocol for use with standard Tris-Glycine SDS-PAGE gels and nitrocellulose membranes. It should give acceptable results in most situations. Optimization for particular situations may require modifying the conditions described.

Buffers and Solutions:

  • Running buffer: 25 mM Tris-HCl, pH 8.3 at RT, 192 mM glycine, 0.1% (w/v) SDS.
  • Transfer buffer: 12 mM Tris-HCl, pH 8.3 at RT, 96 mM glycine, 20% (v/v) methanol. Chill to 2-8oC for best results.
  • Wash Solution:

PBS-Tween: 0.01% (v/v) Tween 20 in Dulbecco's PBS or
TBS-Tween: 20 mM Tris-HCl, pH 7.2-7.4 at RT, 150 mM NaCl, 01% (v/v) Tween 20 or KPl Wash Solution (KPL Cat. No. 50-63-00)

  • Blocking Solution:

2% (w/v) fatty acid-free bovine serum albumin (BSA) in Wash Solution (see above)
or
2% (w/v) nonfat dry milk in Wash Solution
or
KPL Detector Block (fish gelatin based) (KPL Cat. No. 71-83-00)

Protocol:

5.Run gel according to instructions from gel/apparatus manufacturer. Typically, gels are run at 125-200Vconstant voltage. Stop electrophoresis when the dye front is about 1 cm from the bottom of the gel.

6.While the gel is running, soak all fiber pads, filter papers, and transfer membrane in transfer buffer. Be sure that no bubbles are trapped in the filters or fiber pads. (Note: PVDF membranes require prewetting in 100% methanol before soaking in transfer buffer.)

7.After electrophoresis, cut off the bottom right corner of the gel. This will ensure that the gel is oriented correctly in the tranfer apparatus. Equilibrate the gel in transfer buffer for 5-15 min.

8.Assemble the transfer cassette per manufacturer's instructions. Be sure the gel is oriented so that after transfer, the lanes will appear on the membrane in the desired order.

9.Run transfer according to manufacturer's instructions. Common conditions are constant voltage, setting starting voltage at a point which gives a starting current of 200 mA (current will drift down during tranfer). Transfer time is somewhat slower for higher %T gels and for larger proteins, faster for lower %T gels and for smaller proteins. Transfer from a 1 mm-thick minigel in the range of 8-12% acrylamide is usually complete in about 90 minutes. Optimal transfer time should be determined experimentally.

10.After transfer, place the membrane in blocking solution on a rocker platform for at least 30-60 min at RT. Volume should be sufficient to prevent the membrane from drying at any time.

11.Incubate with primary antibody diluted in Blocking Solution on a rocker plate 1 h at RT or overnight at 2-8 oC. Optimal antibody concentration must be determined experimentally. Good starting points are 1-2 g/ml for purified antibodies, or a 1:500 dilution for raw serum or ascites.

12.Wash 3 times for a minimum of 5 minutes each in Wash Solution. Discard each wash.

13.Incubate with secondary antibody conjugate (0.05-1 mg/ml for chromagenic detection, 0.01-0.2 mg/ml for chemiluminescence) in Blocking Solution for 30-60 min at RT on a rocker plate.

14.Wash 3 times for a minimum of 5 minutes each in Wash Solution. Discard each wash.

15.Briefly rinse the membrane in substrate buffer to remove residual detergent--10 sec is sufficient. If this buffer is unavailable, use PBS or Tris without Tween 20.

16.Add substrate. Do not pour substrate solution directly onto membrane.

Chromagenic Detection / Chemiluminescent Detection
13. Develop on rocker for 10-15 min at RT. Do not let background get too dark.
14. Stop reaction as recommended by substrate manufacturer. Alternatively, for peroxidase substrates, stop with deionized water. For phosphatase substrates, stop with either 50 mM Na2-EDTA or deionized water. Air dry membrane.
15. Store membrane long-term wrapped in plastic and out of direct light. / 13. Allow substrate to react for 1 minute.
14. Remove membrane from substrate and drain excess liquid.
15. Lay membrane flat between sheets of clear plastic (transparency films work well).
16. Expose to X-ray film for 1-30 minutes. Do not allow the film to get wet.
17. Develop film according to manufacturer's instructions.

Useful References:

  1. Bollag, D.M., Rozycki, M.D., and Edelstein, S.J. (1996). Protein Methods, Second Edition. New York: Wiley-Liss, 195-227.
  2. Harlow, E. and Lane, D. (1988). Antibodies: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 471-510.