Western Blot Analysis

Detection of specific proteins through Western blot analysis has been an essential tool in biochemical laboratories for many years. This widespread use is owed to the straightforward technique that combines protein separation and antibody-based immunodetection. With appropriate controls, the Western blot may also be used semi-quantitatively, providing useful information on the level of expression of target genes. A diverse set of tools are used throughout this blotting procedure. In this product catalog, we summarize the Western blot workflow while highlighting some defining tools and equipment.

What is the Western blot workflow?

Depending on the experimental goals, Western blot procedures can vary during certain steps. Generally, Western blotting can be broadly summarized as follows:

• Protein extraction - To analyze proteins, they must first be extracted from cells and tissues. This can be done via homogenization, chemical lysis, freeze-thaw and/or sonication. Additional tools such as protein purification kits and protease inhibitors may also be used to improve sample quality and yield.
• Protein gel electrophoresis - Proteins are separated on a gel according to size, often under denaturing conditions. SDS-PAGE is often used, which uses polyacrylamide gels and the SDS detergent. Equipment used in this procedure include electrophoresis chambers, precast gels, and various buffers and reagents.
• Blot transfer - Proteins embedded in the polyacrylamide gel are transferred onto a blotting membrane, such as nitrocellulose or PVDF. This is carried out using an electrophoretic blotter instrument.
• Immunostaining - The membrane is incubated in a solution containing primary antibodies specific to the protein(s) of interest. This is followed by rounds of washing, blocking, and another incubation with secondary antibodies that contain labels for detection. For enzyme labels such as AP or HRP, substrates are also added to finalize the detection of individual protein bands. Fluorescent labels can also be used.
• Gel imaging - To visualize protein bands of interest, specialized gel imagers can be used, which allow multimodal detection modes, such as fluorescence and chemiluminescence. Imagers also often come with analysis software that can measure band intensities, allowing for both qualitative and semi-quantitative Western blot analysis.

Discover useful tools for Western blotting

Browse our catalog to discover many useful Western blotting tools from trusted suppliers ranging from reagent sets to automated instruments. When purchasing supplies, the type of sample to be analyzed should be taken into consideration. Requirements for a small, relatively pure sample will be very different than those for a crude lysate or other complex mixture. Determine also the intended throughput and scale of your team's experiments. Automation-compatible devices may be worthwile for high-volume projects.