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MOLECULAR BIOLOGY: WORKING WITH DNA

EXPRESSION: FUSION PROTEINS

Western Blot Detection

Western Blot Detection Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
Contributor:	The Laboratory of Giovanna Ferro-Luzzi Ames at the University of California, Berkeley
Overview Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
This protocol describes a protein detection process that employs a two-antibody detection system that utilizes alkaline phosphatase conjugated secondary antibodies.
Procedure Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
1. Disassemble the Western Sandwich (see Protocol ID#70). 2. Mark the wells on the Nitrocellulose paper. 3. Cut away the marker lane from the Nitrocellulose paper and place in the Amidoblack Solution for 5 to 10 minutes. 4. Destain the marker lane in Destaining Solution. The marker bands are visualized and can be used to calibrate the rest of the protein blot. 5. Rinse the rest of the Nitrocellulose paper with TNT Buffer. 6. Immerse the membrane in Blocking Buffer and agitate slowly for 1 hr at room temperature. 7. Transfer to the first antibody solution (see Hint #1). Incubate a minimum of 1 hr. 8. Wash three times in TNT Buffer for 10 min each. 9. Transfer to secondary antibody solution (see Hint #2). 10. Incubate with gentle agitation for one hour at room temperature. 11.Wash three times in TNT Buffer for 10 min each. 12. Briefly blot the Nitrocellulose using 3M Whatman paper. 13. Transfer the membrane to the Color Development Solution. A positive reaction will appear within 30 min, and color development will continue for more than 6 hr (see Hint #3). 14. Stop the reaction by discarding the Color Development Solution and add the Stop Color Reaction Buffer.
Solutions Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
Stop Color Reaction Buffer    5 mM EDTA 20 mM Tris, pH 8.0 Color Development Solution    5 mM MgCl2 0.1 M NaCl 1X BCIP Protect solution from light and use within an hour 1X NBT 0.1 M Tris, pH 9.5 BCIP (100X)    20 mg/ml 2-Bromo-3-Chloro-4 Indoyl Phosphate Prepare in 100% Dimethylformamide NBT (100X)    Prepare in 70% (v/v) Dimethylformamide 40 mg/ml Nitrobluetetrazolium Blocking Buffer    20 mM Tris, pH 7.5 0.5 M NaCl 5% (w/v) Powdered Skim Milk 0.05% (v/v) Tween-20 TNT Solution    20 mM Tris, pH 7.5 0.5 M NaCl 0.05% (v/v) Tween-20 Destaining Solution    10% (v/v) Acetic Acid 25% (v/v) 2-Propanol Amidoblack Solution    10% (v/v) Acetic Acid 25% (v/v) Isopropanol 0.1% (w/v) Amidoblack 10B
BioReagents and Chemicals Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
Sodium Chloride Nitrobluetetrazolium Milk, Powdered Skim Amidoblack 10B 2-Bromo-3-Chloro-4 Indoyl Phosphate Dimethylformamide (DMF) Tris 2-Propanol Tween-20 Acetic Acid EDTA Isopropanol Magnesium Chloride
Protocol Hints Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
1. The antibody concentration needs to be optimized for each antibody. The general range for primary antibodies used on Western blots ranges from undiluted to 1:10,000. 2. The antibody concentration needs to be optimized for each antibody. The general range for secondary antibodies used on Western blots ranges from 1:1000 to 1:20,000. 3. The optimum time for color development is dependent on the antibody specificity and needs to be adjusted empirically for optimum signal to noise ratio.