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ECM Kit (goat IgG) for Western Blotting (OKBB00068)

  • Catalog#: OKBB00068
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Application:
ELISA
Kit Component:
1. Blocking buffer: 10g protein dry powder (skimmed milk powder). 2. HRP‐conjugated rabbit anti‐goat IgG: Affinity purified antibody. 2000‐10000×. 3. Enhanced chemiluminescent chromogenic reagent, containing: Chromogenic reagent A: 5ml. 20× ; Chromogenic reagent B: 5ml. 20×.
Kit Detection Method:
Colorimetric, OD450 nm
Datasheets/Manuals:
Printable datasheet for OKBB00068
Reconstitution and Storage:
4C for 1 year.
Preparation:
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 the membrane: make the blocking solution by dissolving 2g protein dry powder (Component 1) in 100 ml Diluent Buffer and use within 24 hours; completely submerge the membrane in the blocking solution and incubate at 20‐37C for 1‐2 hours or at 4C overnight with agitation. 4. Wash membrane once for 10 minutes with Wash Buffer. 5. Preparation of Antibody Diluent Solution: Preparation of Antibody Diluent Solution: add 2g protein dry powder and 1g BSA into 100ml of Diluent Buffer. Dilute primary antibody (user‐supplied) and secondary antibody (Component 2) with the Antibody Diluent Solution.6. Incubate the membrane in diluted primary antibody at 20‐37C for 2 hours or at 4C overnight with agitation. 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 10 minutes each.8. Incubate the membrane with diluted secondary antibody at 20‐37C for 90 minutes or at 4C overnight. 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 chemiluminescent reagent to detect the bands. Add one drop of each chromogenic reagent A, B (Component 3) into 1ml of distilled water and mix thoroughly. Add the resulting solution to the membrane and develop at room temperature until bands appear (usually 1‐5 minutes).11. Gently blot the edge of the membrane on a piece of paper to remove excess chromogenic reagent. Wrap the membrane in plastic wrap in order to keep X‐ray film dry. Develop the film by exposure between 30s to 5 minutes in a dark room.
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Notes: "Goat 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 goat. Every kit has sufficient reagents for 800 cm2 of membranes.

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