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1. Nativepage Novex Bis-tris Gel System User Manual Enhanced
2. Nativepage Novex Bis-tris Gel System User Manual Sample
3. Nativepage Novex Bis-tris Gel System User Manual Template
4. Nativepage Novex Bis-tris Gel System User Manual 2017 3 Pdf

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Abstract

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.

Iomega screenplay director hd media player user manual. 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.

Protocol

Gel electrophoresis

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 tips

Nativepage Novex Bis-tris Gel System User Manual Enhanced

• In the Invitrogen NuPAGE Bis-Tris discontinous buffer system, the electrophoretic mobility of the proteins and the subsequent separation range of the gel is dependent on two factors: i) the acrylamid concentration of the gel (with greater acrylamide concentration resulting in better resolution of lower molecular weight proteins) and ii) the trailing ion of the running buffer, MOPS or MES. To choose the appropriate combination for the separation range that you wish to achieve, refer to the Gel Migration chart¹. The thickness of the gel and number of wells will depend on the volume and the quantity of samples that you plan to load, respectively.

• When you load your samples into wells of the gel, at least one lane is reserved for a molecular weight marker (or ladder). These protein standards are commercially available from several companies and typically consist of a mixture of stained proteins having defined molecular weights, so as to form visible bands that allow you to follow the migration progress. Pick a range of molecular weights that is compatible with your gel resolution.

• To achieve a nice and even migration pattern we recommend loading ALL the wells of your gel with a similar volume of sample or 1X LDS sample buffer.

Transfer

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):

• After transferring the proteins, place the membrane in an incubation tray (proteins facing up).

• Add enough Ponceau S Staining to cover the membrane and incubate at least 30 seconds with gentle agitation.

• Rinse membrane with distilled water until the background is clear.

• Destain the membrane with running distilled water for 2-3 minutes.

• Place the membrane in blocking solution (see below).

Nativepage Novex Bis-tris Gel System User Manual Sample

Technical tips:

• We recommend labeling your membrane with a pencil before protein transfer in order to denote the side in which the proteins were exposed and the orientation of your samples.

• Both Ponceau S staining and the molecular weight markers may be used to cut the membrane after transfer for probing the resulting parts with different antibodies

Nativepage Novex Bis-tris Gel System User Manual Template

Immunodetection:

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 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.

Nativepage Novex Bis-tris Gel System User Manual 2017 3 Pdf