Department of Physiology and Biophysics, University of California, Irvine (UCI)
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Penna, A., Cahalan, M. Western Blotting Using the Invitrogen NuPage Novex Bis Tris MiniGels. J. Vis. Exp. (7), e264, doi:10.3791/264 (2007).
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. In this method, a target protein is detected with a specific primary antibody in a given sample of tissue homogenate or extract. Protein separation according to molecular weight is achieved using denaturing SDS-PAGE. After transfer to a membrane, the target protein is probed with a specific primary antibody and detected by chemiluminescence.
Since its first description, the western-blotting technique has undergone several improvements, including pre-cast gels and user-friendly equipment. In our laboratory, we have chosen to use the commercially available NuPAGE electrophoresis system from Invitrogen. It is an innovative neutral pH, discontinuous SDS-PAGE, pre-cast mini-gel 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).
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. In our laboratory, we have obtained good and reproducible results for various biochemical applications using this western-blotting method.
Technical note: Before starting the procedure, have your protein samples ready.
During this first step, the proteins in the sample are separated according to their molecular weight using denaturing polyacrylamide gel electrophoresis (PAGE). The NuPAGE® LDS Sample Buffer loaded with Lithium Dodecyl Sulfate (LDS) maintains polypeptides in a denatured state once the protein sample has been heated at 70°C for 10 minutes. A strong reducing agent is used in conjunction to remove secondary and tertiary structure (DTT, to break disulfide bonds). In addition, sample proteins become covered in the negatively charged LDS and therefore move through the acrylamide mesh of the gel toward the positively charged electrode. This allows their separation according to molecular weight (measured in kilo Daltons, kDa).
Detailed step-by-step protocols for the sample preparation and the PAGE procedure can be found on the Invitrogen website2, or in the NuPAGE technical guide3.
Technical Note: Before beginning the transfer step, prepare the transfer buffer (1X with 10% methanol) and pre-cool to 4°C in a cold room.
In order to make the proteins accessible to antibody detection, they are transfered by electroblotting from the gel onto a nitrocellulose membrane. Protein binding is based upon hydrophobic interactions, as well as charge interactions between the membrane and protein.
(Polyvinylidene fluoride (PVDF) membrane can also be used as an alternative. In this case, the PVDF membrane needs to be pre-wet in methanol at least 30 seconds before use.)
A 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.
As a result of this process, the proteins are exposed on a thin surface layer and ready for detection. 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.
Ponceau S staining procedure (optional):
Technical Note: This immunodetection procedure is provided as a guideline only. Optimization may be required for each antibody; specific information can be found in most data-sheets of the commercially available antibodies (e.g., working dilution, incubation time, etc.).
During this last process, the target protein will be detected using a specific antibody and will appear as a band on the film. The position of the band is dependent of the molecular weight of the target protein, whereas the band intensity depends on the amount of target protein present.
Typically, this is achieved after three substeps:
A generic step-by-step procedure is provided below. Other detailed procedures can be found in the ECL Plus Western Blotting Detection Reagents instruction manual5 or in most data-sheet accompanying the commercially available antibodies. Incubation time, antibody dilution, and blocking and wash solutions have to be empirically optimized for each antibody.
Now that you have exposed your film, you will realize that in practical terms, not all Westerns reveal protein as one nice single band. Additional bands may also appear due to the non-specific binding of both primary and secondary antibodies. This background signal can be reduced by optimizing the immunodetection procedure. 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.
After 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). 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). Determine the regression line of the standard curve to obtain values for slope and y-intercept. The unknown molecular weight (size) of your target protein is estimated using its Rf and the following modified equation:
log molecular weight = (slope)(mobility or Rf of the target protein) + y-intercept
(see reference 6 for detailed instructions).
Expression 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. This so called "loading control" should not change between samples and is revealed using a specific primary antibody. The image may be further analyzed by densitometry to evaluate the relative amount of protein staining and quantify the results in terms of optical density.
The procedure presented here uses a Bis-Tris gel with MES running buffer as an example of denaturing PAGE using the Invitrogen NuPAGE Novex electrophoresis system. In addition, this pre-cast gel system allows protein separation under denaturing or non-denaturing conditions as well as accomodates a wide range of molecular weights (from 1-200 kDa for the Bis-Tris gels to 36-400 kDa for the Tris-Acetate gels). Depending on your experiment, you may choose to follow only part of the western-blotting technique described here and use direct gel staining procedures (e.g., silver or Coomassie staining) or an alternative immunodetection method (e.g., colorimetric or fluorescent).
We routinely use the Invitrogen NuPAGE Novex electrophoresis system in our laboratory for various applications, some examples of which can be found in references 7-9.