Western blotting (WB) or protein blotting is a technique used to detect specific proteins in a given sample. During the process, the proteins resolved on the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) gel are transferred to adsorbent membrane supports under the action of an electric current. Then, the membrane is incubated with a labeled antibody specific for the protein of interest. The unbound antibody is washed away, leaving only the antibody bound to the target protein. When the antibody binds to the appropriate substrate, a detectable signal will produce. Immunodetection is a powerful tool for detecting and characterizing a multitude of proteins, especially those with low abundances. Compared to gels, wet membranes are pliable and easy to handle, and the proteins immobilized on the membranes have easy access to different ligands. Transfer analysis requires only a small number of reagents, and transfer patterns can be stored for a long time before use. In addition, the same protein transfer can be used for multiple successive analyses. The efficient transfer of proteins depends mainly on the nature of the gel, the molecular mass of the protein being transferred, and the membrane used. Using thinner gels, the transfer becomes more complete and faster. However, the use of ultra-thin gels may lead to handling problems. Proteins with high molecular mass are poorly blotted following SDS PAGE, resulting in a low level of detection by immunoblotting.
Western blotting equipment is used to transfer and identify specific proteins in samples, reveal protein modifications, and give semi-quantitative estimates of their concentrations. Western blotting equipment includes all apparatus required to transfer proteins from the gel to the membrane and subsequent processing steps. Protein transfer can be performed by electroblotting with wet, semi-dry, or dry transfer systems onto nitrocellulose (NC) and polyvinylidene fluoride (PVDF) membranes. The PVDF membrane has higher protein binding capacity than that of the NC membrane and therefore are more suitable for detecting proteins with low abundance. The transfer protocol depends on the protein type, gel thickness, and membrane type. After transfer, proteins are probed, stained, washed, and detected. The assay may require cross-linking or hybridization to immobilize proteins, trays for washing, staining, and staining membranes, and holders to mount gels and blots. Automated equipment can shake, block, hybridize, wash blots, and even recover primary antibodies, thus freeing up time for other important laboratory work. Finally, proteins can be detected and imaged using gel documentation and imaging systems.
Considerations for selecting western blotting equipment include the cost of primary and secondary antibodies, proteins of interest, time required for transfer, throughput, ease of cleanup, flexibility of protocol modifications, and type of assay required. Amerigo Scientific offers easy-to-use western blotting equipment such as imaging systems, transfer systems, and blotting systems.
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