BIO RAD WESTERN BLOT GEL HOW TO
BIO RAD WESTERN BLOT GEL SERIES

This background signal can be reduced by optimizing the immunodetection procedure. Additional bands may also appear due to the non-specific binding of both primary and secondary antibodies. Now that you have exposed your film, you will realize that in practical terms, not all Westerns reveal protein as one nice single band. The uniformity and overall effectiveness of transfer of protein from the gel to the membrane can be checked by the reversible Ponceau S dye membrane staining. In this case, the PVDF membrane needs to be pre-wet in methanol at least 30 seconds before use.)Ī detailed step-by-step protocol of the transfer procedure can be found in reference 4 if you use the Bio-Rad Mini Trans-Blot Electrophoretic Transfer Cell, or in references 2-3 if your lab is equipped with the Invitrogen’s XCell II™ Blot Module.Īs a result of this process, the proteins are exposed on a thin surface layer and ready for detection. (Polyvinylidene fluoride (PVDF) membrane can also be used as an alternative. Protein binding is based upon hydrophobic interactions, as well as charge interactions between the membrane and protein. In order to make the proteins accessible to antibody detection, they are transfered by electroblotting from the gel onto a nitrocellulose membrane. Technical Note: Before beginning the transfer step, prepare the transfer buffer (1X with 10% methanol) and pre-cool to 4☌ in a cold room. In our laboratory, we have obtained good and reproducible results for various biochemical applications using this western-blotting method.

BIO RAD WESTERN BLOT GEL HOW TO

The procedure described in this video article utilizes the Bis-Tris discontinuous buffer system with 4-12% Bis-Tris gradient gels and MES running buffer, as an illustration of how to perform a western-blot using the Invitrogen NuPAGE electrophoresis system. This system presents several advantages over the traditional Laemmli technique including: i) a longer shelf life of the pre-cast gels ranging from 8 months to 1 year ii) a broad separation range of molecular weights from 1 to 400 kDa depending of the type of gel used and iii) greater versatility (range of acrylamide percentage, the type of gel, and the ionic composition of the running buffer). It is an innovative neutral pH, discontinuous SDS-PAGE, pre-cast mini-gel system. In our laboratory, we have chosen to use the commercially available NuPAGE electrophoresis system from Invitrogen. Since its first description, the western-blotting technique has undergone several improvements, including pre-cast gels and user-friendly equipment. After transfer to a membrane, the target protein is probed with a specific primary antibody and detected by chemiluminescence. Protein separation according to molecular weight is achieved using denaturing SDS-PAGE. In this method, a target protein is detected with a specific primary antibody in a given sample of tissue homogenate or extract. The Mini-PROTEAN Tetra Cell can be configured for use with 1–4 precast or handcast gels in different thicknesses.Western Blotting (or immunoblotting) is a standard laboratory procedure allowing investigators to verify the expression of a protein, determine the relative amount of the protein present in different samples, and analyze the results of co-immunoprecipitation experiments. Requires a PowerPac Basic or comparable power supply.Leak-free electrophoresis and gel casting.Indestructible molded polycarbonate construction.Runs 1–4 mini polyacrylamide gels in 30 min.

BIO RAD WESTERN BLOT GEL SERIES

Protein Expression and Purification Series.Comparative Proteomics Kit I: Protein Profiler Module.Use the Mini-PROTEAN Tetra cell to teach protein fingerprinting, sample purity analysis, and downstream western blotting techniques in independent projects or with the following educational kits: An indispensable piece of equipment in research laboratories, the durable and easy-to-assemble Mini PROTEAN Tetra cell is also well-suited for use in classroom laboratories. Bio-Rad’s Mini-PROTEAN Tetra cell is a mini-format, vertical polyacrylamide gel electrophoresis system that provides rapid, high-resolution separation of protein (or DNA) mixtures by SDS-PAGE or native PAGE.

Bio rad western blot gel