This so called "loading control" should not change between samples and is revealed using a specific primary antibody. (see reference 6 for detailed instructions).Įxpression level approximations are taken by comparing the band intensity of the target protein to that of a structural protein (e.g., tubulin or actin) or a housekeeping gene product such as GAPDH. Log molecular weight = (slope)(mobility or Rf of the target protein) + y-intercept The unknown molecular weight (size) of your target protein is estimated using its Rf and the following modified equation: Determine the regression line of the standard curve to obtain values for slope and y-intercept. Relative mobility (Rf) is the term used for the ratio of the distance the protein has moved from its point of origin (top of the gel) relative to the distance the tracking dye or a low molecular weight marker has moved (the gel front). In addition, an appropriate control (e.g., untransfected cells, siRNA-treated cells, etc ) will be useful to determine the specificity of your antibody and the exact location of the target protein on the membrane.Īfter marking on the film the position of the stained protein standard bands from the membrane, plot the log of each molecular weight of the protein standards (y-axis) against their corresponding relative mobility (x-axis). 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. 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. 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.
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