Blotting and detection

What is Western blotting?

Western blotting is a research method that uses antibodies to identify target proteins in a cell lysate sample. Blotting is one of the most common analytical methods for detecting and quantifying proteins and nucleic acids.

Western blot leverages three elements to achieve separation:

• size or molecular weight
• protein transfer to solid supports
• marking the target protein using antibodies.

Western blot protocols usually entail protein separation via SDS polyacrylamide gel electrophoresis (SDS-PAGE), then transfer to a polyvinylidene difluoride (PVDF) or nitrocellulose membrane. Once proteins are transferred to the solid support, they are stained for easy visualization and identification through mass spectrometry or immunodetection. For each of these steps, different Western blot materials are used.

During Western blotting immunodetection, the proteins of interest are identifiable through their binding to specific antibodies. Primary antibodies combined with an HRP- or AP-conjugated secondary antibody facilitate colorimetric detection.

Applications of Western blotting

Researchers and biomanufacturers use Western blotting for:

• detecting the presence of specific proteins
• determining the extent of post-translational modifications
• confirming protein expression in cloning
• analyzing protein and biomarker expression
• mapping of antibody epitope

Using suitable Western blot materials and protocols ensures your blotting techniques' high sensitivity and speed.

Western blot materials

Below are the materials commonly used in Western blot protocols:

• nitrocellulose or PVDF transfer membranes
• transfer buffer
• wash buffer
• blocking buffer
• incubation trays and containers
• primary antibodies
• secondary antibodies
• chemiluminescent HRP substrate

Western blot materials FAQs

Below are answers to a few frequently asked questions about western blot materials:

What reagents are used in Western blotting?
Reagents used in Western blot protocols include:

• PAGE buffers
• protein markers
• blotting membranes
• protein gel staining kits
• blocking buffers
• ECL substrates
• Western blot imagers

What paper is used in Western blotting?
Western blotting uses Western blot filter papers and pads to facilitate the efficient transfer of proteins. These cotton cellulose fibers support use in other blotting applications like Northern and Southern blot techniques.

What types of membranes are used in Western blotting?
The two main types of Western blotting membranes are PVDF and nitrocellulose membranes. These membranes feature high binding capacity, large surface area to volume ratio, and extended storage of molecules. They are easy to use and come in different pore sizes.

• Nitrocellulose membranes: For Western blotting, nitrocellulose membranes are a common choice because of their high protein binding affinity of 80–100 g/cm2. They can immobilize a wide range of proteins and are compatible with a wide range of detection modalities. Hydrophobic interactions are the most common method of protein immobilization. Protein immobilization is enhanced by an electrophoretic transfer in the presence of high salt and low methanol concentrations. High binding efficiency and irreversibility are the hallmarks of these membranes.
• PVDF membranes: Nucleic acids and proteins have a high propensity for attaching to PVDF transfer membranes, which are very hydrophobic. It is necessary to soak them with ethanol or methanol first and then place them in a transfer solution. PVDF can withstand more reprocessing rounds than nitrocellulose because it is more durable.