Western Blotting Goat IgG DAB Chromogenic Reagent Kit (Yellow)

DAB is the chromogenic substrate of peroxidase. The reaction product is a brown precipitate insoluble in water, dimethylbenzene or alcohol, which makes DAB suitable for color development reaction in western blotting. Boster DAB Chromogenic Reagent kit is extremly senstive and has a high signal-to-noise ratio.

˙ Nitrocellulose or PVDF membrane.
˙ Diluent Buffer (for preparation of blocking reagent and antibody dilutions): Add 2.42g Tris, 9g NaCl,
850?900ul pure acetic acid into 1000ml distilled water, adjust pH to 7.2?7.6.
˙ Wash Buffer: Add 0.5 ml of TWEEN 20 into 1000 ml of diluent buffer.
˙ Primary antibody: This kit applies to the primary antibodies raised from goat.

Assay Procedure
1. Run protein sample and molecular weight standard through polyacrylamide gel electrophoresis (PAGE).
2. Transfer the protein sample to a nitrocellulose membrane or PVDF membrane.
3. Block membrane: Immerse the membrane in blocking buffer and incubate at 20-30C for 1.5-2 hours or at 4C overnight with agitation.
4. Wash membrane once for 10 minutes in Wash Buffer.
5. Incubate membrane with primary antibody: Dilute primary antibody in Diluent Buffer. Incubate membrane with primary antibody solution at 20-30C for 2 hours or at 4C overnight with agitation. Follow the antibody manufacturer’s recommendations for optimized concentration.
6. Wash membrane in Wash Buffer with gentle agitation, 3 times for 10 minutes each.
7. Incubate the membrane with diluted secondary antibody at 20-30C for 90 minutes or at 4C overnight. Secondary antibody dilutions typically range from 1:2000-1:10000. Optimal secondary antibody dilutions must be determined empirically.
8. Wash membrane in Wash Buffer with gentle agitation, 4 times for 5 minutes each.
9. Chemiluminescent Detection: Add 50ul chromogenic reagent A, B and C into 2 ml of distilled water and mix well. Add the working solution onto the membrane and incubate at room temperature until bands appear (usually 1-5 minutes). Wash the membrane with distilled water to stop the reaction.
10. Observe the bands and take pictures.