A western blot is a method in molecular biology/biochemistry/immunogenetics to detect protein in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate denatured proteins by mass. The proteins are then transferred out of the gel and onto a membrane, where they are “probed” using antibodies specific to the protein. As a result, researchers can examine the amount of protein in a given sample and compare levels between several groups.
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Secondary Antibody
Tag Antibody
Loading Control Antibody
Isotype Control Antibody
Immunological Detection of Antigens
Handling of Antibody and Antibody-Enzyme Conjugate Reagents
Reagent Preparation
Immunoblotting is typically used to determine the amount (dot blot) and molecular weight (western blot) of an antigen present in a complex mixture. The highly sensitive procedure below is suggested. All incubations are at room temperature.
This procedure is for nitrocellulose or PVDF membranes. Do not touch the membrane! Wear gloves. PVDF membranes must be pre-wet with 100% methanol before equilibrating in transfer buffer. Nylon membranes may be used, however, they are more difficult to block. To block nylon membranes use buffer without Tween-20, replace BSA with 10% nonfat dry milk and block for several hours to overnight at 4 degrees C. When selecting membranes, the specifications of the particular imaging systems with their available filters must be taken into consideration. Some membranes may exhibit auto fluorescence at certain wavelengths.
Western blots allow investigators to determine the molecular weight of a protein and to measure relative amounts of the protein present in different samples.
1. Transfer and immobilize antigen on nitrocellulose or PVDF membrane.
2. Block by immersing the membrane in TTBS. Use just enough solution to cover the membrane. Never let the membrane become dry during the procedure. Incubate for 30 minutes with gentle agitation. The addition of 1.0% BSA to the blocking solution increases signal-to-noise ration over the use of TTBS aloe. Some antigens and antibodies may be eluted in the presence of Tween-20. If this occurs, replace the Tween-20 with 1.0% BSA in all TTBS solutions and repeat the experiment.
3. Transfer the membrane to diluted solution of primary antibody in TTBS. The appropriate dilution should be determined by trial and error. Serial ten fold dilutions starting at 1:10 are suggested. Incubate for 30 minutes with gentle agitation.
4. Wash with 3 changes of TTBS for 5 minutes each with gentle agitation.
5. Transfer the membrane to a dilute solution of biotinylated secondary antibody in TTBS. Confirm reagent specificity for primary antibody. Incubate for 30 minutes with gentle agitation.
6. Repeat step 4.
7. Transfer the membrane to a solution of peroxidase conjugated streptavidin appropriately diluted in TTBS. Incubate at 30 minutes with gentle agitation.
8. During the above incubation, prepare the enzyme substrate. Use DAB for a dark brown color or TMB for a bright blue color.
9. Wash as in step 3.
10. Transfer the membrane to the substrate solution. Incubate until color develops sufficiently (usually 2-20 minutes).
11. Wash with 2 changes of water for 5 minutes each with gentle agitation. Allow membrane to dry and store in the dark.
Western Blot or immunoblot is a workhorse immunoassay for most labs, used to demonstrate the presence or absence of important proteins, detect post-translational modifications, diagnosis of disease and more. Not all antibodies work well in a western blot assay, due to the fact that the protein is denatured and the epitope (antibody binding site) of the protein of interest may not be accessible. Specific screening may be required to detect antibodies that are compatible with western blotting and optimization of the assay conditions to maximize their performance.
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