Name: western blotting for protein detection
Uploaded: 2023-04-30T11:58:31.000Z
Duration: 1 min 15 s
Channel: JoVE

Western blotting is an analytical technique used to detect the protein of interest from a protein mixture.
It can help identify protein isoforms and post-translational protein modifications. Further, it can ascertain target proteins following drug treatment.
This method builds on the SDS-PAGE that separates proteins based on the charge to mass ratio.
These proteins are transferred to a nitrocellulose membrane, typically under an electric current.
The negatively charged proteins move towards the anode and are transferred to the membrane retaining their original position.
The membrane is then incubated with a primary antibody, specific to the protein of interest. A prior incubation in a blocking buffer prevents the binding of antibodies to the membrane.
Next, the secondary antibody is tagged with reporter enzymes, like horseradish peroxidase or a fluorophore, which binds to the primary antibody.
When incubated with an appropriate substrate, these reporter enzymes produce color or light, or emit fluorescent signals allowing the detection of the specific protein in the mixture.

western blotting

Western Blotting

Western blotting is an analytical technique for protein identification. It has various applications in immunology and medicine, including detecting diseases like bovine spongiform encephalopathy, mad cow disease, and human and feline immunodeficiency virus from biological samples.
The technique begins with separating proteins from the sample using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by protein transfer, immunoblotting, and finally, protein detection.
The proteins from the polyacrylamide gel are transferred to specialized membranes like nitrocellulose (NC) or polyvinylidene difluoride (PVDF). Compared to NC, PVDF has higher durability and protein binding capacity and can be re-probed.
The protein transfer is either by electric current application (electroblotting) or capillary action. In electroblotting, the hydrophobic interactions transfer the negatively charged proteins from the gel and trap them on the membrane. In the other method, a filter paper placed on the gel soaks the buffer by capillary action, simultaneously drawing the proteins from the gel and transferring them to the membrane. To ensure the quality of transferred bands, non-specific protein stains like Ponceau S allow for a reversible check.
The next step, immunoblotting, involves treating the membrane with a blocking buffer like 3-5% Bovine serum albumin or non-fat dried milk. These buffers reduce the chance of non-specific binding of the primary antibody to the membrane. The primary antibodies can either be polyclonal or monoclonal and bind to the protein bands on the membrane through their Fab region. Next, the added secondary antibodies bind and attach to the Fc region of the primary antibodies. The secondary antibodies are tagged with enzymes which, upon suitable substrate addition, produce detectable colorimetric or chemiluminescent signals. Further, densitometry techniques can quantify the optical density of these imaged bands, corresponding to the abundance of proteins in the sample.
The expected result for a western blot can be affected by various factors like erroneous sample loading, inaccurate transfer with trapped air bubbles, and the use of non-specific antibodies. Also, inappropriate primary and secondary antibody concentrations and contaminated buffers can affect the quality of bands and their visualization.

Western blotting is an analytical technique for protein identification. It has various applications in immunology and medicine, including detecting ...

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Cell Biology

Unit 1

Analyzing Cells and Proteins

KEY TERMS AND CONCEPTS

western blotting for protein detection

Western blotting is a protein detection method used in biology and medicine. It helps identify proteins in biological samples and can support disease detection, including bovine spongiform encephalopathy, human immunodeficiency virus, and feline immunodeficiency virus.

The process starts with SDS-PAGE, which stands for sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This step separates proteins in a sample before they are moved to a membrane. The proteins are then transferred to nitrocellulose (NC) or polyvinylidene difluoride (PVDF) membranes. PVDF is more durable than NC, binds proteins well, and can be used again after re-probing.

Protein transfer can happen in two ways. In electroblotting, an electric current helps move the negatively charged proteins from the gel to the membrane through hydrophobic interactions. In the capillary method, filter paper draws up buffer by capillary action and pulls the proteins from the gel onto the membrane at the same time. A reversible stain such as Ponceau S can be used to check that the bands transferred properly.

After transfer, the membrane is blocked with a buffer such as 3-5% bovine serum albumin or non-fat dried milk. Blocking reduces non-specific binding of the primary antibody to the membrane. The primary antibody may be polyclonal or monoclonal, and it binds the target protein band through its Fab region. A secondary antibody then binds to the Fc region of the primary antibody and carries an enzyme that makes the signal visible after substrate is added.

The final signal can appear as a colorimetric or chemiluminescent band. Densitometry can then measure the optical density of the image to estimate protein abundance in the sample. Several problems can affect the result, including incorrect sample loading, poor transfer caused by trapped air bubbles, non-specific antibodies, wrong primary or secondary antibody concentrations, and contaminated buffers.

Western Blotting for Protein Detection

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Western blotting is a protein detection method used in biology and medicine. It helps identify proteins in biological samples and can support disease detection, including bovine spongiform encephalopathy, human immunodeficiency virus, and feline immunodeficiency virus. The process starts with SDS-PAGE, which stands for sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This step separates proteins in a sample before they are moved to a membrane. The proteins are then transferred to...

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key terms and concepts

Learn how western blotting separates proteins, transfers them to a membrane, and uses antibodies to detect disease-related protein bands in samples.

Western Blotting for Protein Detection | JoVE K12

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Western Blotting for Protein Detection

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