- Kit Component:
- 1. Blocking Reagent: 2×10g protein dry powder. 2. Concentrated antibody diluent solution: 20 ml/bottle. 10×. 3. Polymeric peroxidase‐conjugated goat anti‐rabbit IgG: 0.2 ml. 200‐400×. 4. DAB chromogenic reagent, containing: Chromogenic reagent A: DAB concentrated solution, 3ml. 40×. Chromogenic reagent B: H2O2 concentrated solution, 3ml. 40×. Chromogenic reagent C: TBS concentrated buffer, 3ml. 40×.
- Kit Detection Method:
- Colorimetric, OD450 nm
- Printable datasheet for OKBB00113
- Reconstitution and Storage:
- ‐20C for 1 year. DAB reagent should be protected from light.
- 1. Run protein sample and molecular weight standard through polyacrylamide gel electrophoresis (PAGE).
FOR RESEARCH USE ONLY. NOT FOR DIAGNOSTIC AND CLINICAL USE.
Product Information Sheet www.immunoleader.com
2. Transfer the protein sample to a nitrocellulose membrane or PVDF membrane.
3. Block the membrane: make the blocking solution by dissolving 2g protein dry powder (Component 1) in 100 ml Diluent Buffer; completely submerge the membrane in the blocking solution and incubate at 20‐37C for 1‐2 hours.
4. Wash membrane once for 5 minutes with Wash Buffer.
5. Preparation of Antibody Diluent Solution: add 1g protein dry powder and 10ml of concentrated antibody diluent solution (Component 2) into 90ml of Diluent Buffer. Dilute primary antibody (user‐supplied) and secondary antibody (Component 3) with the Antibody Diluent Solution.
6. Incubate the membrane in diluted primary antibody at 20‐37C for 2 hours. Follow the antibody manufacturer’s recommendations for best concentration. In case of absence of the specific bands or weak positive staining, increase the concentration of the primary antibody; in case of presence of non‐specific bands, decrease the concentration of the primary antibody.
7. Wash the membrane by agitating it in Wash Buffer, 3 times for 5 minutes each.
8. Incubate the membrane with diluted secondary antibody at 37C for 90 minutes. The user may adjust the dilution based on actual staining.
9. Wash the membrane by agitating it in Wash Buffer, 4 times for 5 minutes each.
10. Use DAB chromogenic reagent to detect the bands. Add one drop of each chromogenic reagent A, B and C (Component 4) into 2ml of distilled water and mix thoroughly. Add the resulting solution to the membrane and develop at room temperature until bands appear (usually 1‐30 minutes). Wash the membrane with distilled water to stop the reaction.
11. Observe the bands and take pictures.
- Notes: - "Rabbit IgG" refers to the origin of animal species of the primary antibody, not the origin of the specimen. This kit applies to the primary antibodies raised from rabbit.
- Store the kit at 4C for frequent use, or at ‐20C for infrequent use. The shelf life at ‐20C is one year.
- Chromogenic reagent A should be stored at ‐20C. If crystal appears, fully dissolve it before use.
- Chromogenic working solution should be freshly prepared.