To describe the procedure to develop western blot antigen strips for analysis of antigens or antibodies.


Western Blotting of Proteins, Clinical Biotechnology (1989); 1(1):43-49


  • 1X PBST20 (from SOP-103)
  • RMS-142  Methanol
  • SOP-105  Diluent Buffer for HRP Conjugates
  • RMS-162  Normal Goat Serum
  • SOP-120  Heat Inactivation of Animal Serum
  • SOP-141  Preparation of 5M NaCl
  • RMS-178  Streptavidin-AP
  • RMS-179 Gt anti-Human IgG-AP
  • RMS-180  Gt anti-Mouse IgG-AP
  • RMS-181  BCIP/NBT Reagent Kit


  • Rotator (LabLine # 1314 or equivalent)
  • BioIce container (frozen with DI water at -20°C) BioRad 170-3934
  • Strip incubation reservoirs (BioRad 170-3902)
  • Plate Incubator (Boekel # 260700 or equivalent)
  • Tube Rocker (Nutator # 421105 or equivalent)
  • Vacuum Aspirator - Remove the cotton plug from a 10 ml pipette and attach the pipette to a length of 1/4" I.D. plastic tubing.  Attach the other end of the tubing to one outlet of a two outlet sealed reservoir and attach a vacuum source to the other outlet.  Use this to remove samples and washes from the incubation trays.



  1. Prepare a sufficient volume of  Antibody Diluent for primary and secondary antibody incubations by  mixing one volume Diluent Buffer (SOP-105), 1/10 volume 0.5M NaCl (SOP-140) and 1/20 volume Heat inactivated normal goat serum (RMS-162 + SOP-120).
  2. If using human serum/plasma antibody samples, dilute 1:100 by adding 10 µl serum/plasma to 990 µl antibody diluent.
  3. If using monoclonal antibodies, dilute appropriately in antibody diluent using the Certificate of Analysis dilution data as a guide.
  4. Place antigen strips in strip reservoir and wet with 1 ml 50% methanol.  Aspirate and add 1 ml per strip antibody diluent.  Incubate for five minutes.  Aspirate and add 1 ml per strip diluted antibody samples .
  5. Incubate at 37°C on rotator for 60 minutes.
  6. Aspirate and wash with about 2 ml PBST20 per strip 1X 10 seconds, 2X 5 minutes.
  7. Place the MW marker strip in a 15 ml screw-cap centrifuge tube with 2 - 3 ml 50% methanol.  After strip is wet, decant and rinse with 10 ml antibody diluent.  Decant and add another 10 ml antibody diluent and add a 1:1,000 dilution of Streptavidin-AP (10 µl).  Place on tube rocker for 30 minutes at room temperature.
  8. Add 1 ml per strip of a 1:5,000 dilution of Gt anti-Human IgG (H&L)-AP in antibody diluent.  Incubate at 37°C on rotator for 30 minutes.
  9. Aspirate and wash as in step 5.26.  Rinse marker tube in the same way with 10 ml PBST20 per wash.
  10. Rinse 1X 5 minutes with DI water.  Two ml per strip and 10 ml in the marker tube. Do not aspirate.
  11. Make sufficient BCIP/NBT solution for 1 ml per strip and 5 ml for the marker tube.  Add 1/100 volume Reagent A and Reagent B to the AP Substrate buffer.
  12. Transfer the strips to a dedicated substrate incubation tray.  Add one ml per strip BCIP/NBT solution.  Decant the marker tube and add 5 ml BCIP/NBT solution. Incubate at room temperature for 15 minutes while rotating the strips and rocking the marker tube.
  13. Aspirate and rinse with 2 ml DI water per strip and 10 ml DI for the marker tube.  After 5 - 10 minutes, remove the strips and markers to a piece of transfer filter paper for drying overnight.