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
- 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).
- If using human serum/plasma antibody samples, dilute 1:100 by adding 10 µl serum/plasma to 990 µl antibody diluent.
- If using monoclonal antibodies, dilute appropriately in antibody diluent using the Certificate of Analysis dilution data as a guide.
- 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 .
- Incubate at 37°C on rotator for 60 minutes.
- Aspirate and wash with about 2 ml PBST20 per strip 1X 10 seconds, 2X 5 minutes.
- 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.
- 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.
- Aspirate and wash as in step 5.26. Rinse marker tube in the same way with 10 ml PBST20 per wash.
- Rinse 1X 5 minutes with DI water. Two ml per strip and 10 ml in the marker tube. Do not aspirate.
- 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.
- 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.
- 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.