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Western Blot Illustrated Assay: How to Run a Western Blot

This How To Guide Will Cover

Western Blot Theory Overview

Antibodies bind to specific sequences of amino acids, known as the epitope. Because amino acid sequences are different from protein to protein, antibodies can recognize specific proteins among a group of many. Therefore, a single protein can be identified in a cell lysate that contains thousands of different proteins and its abundance quantified through western blot analysis. First, proteins are separated from each other based on their size. Second, antibodies are used to detect the protein of interest. Finally, a substrate that reacts with an enzyme is used to view the antibody/protein complex.

Sample Preparation

Western blot sample preparation diagram involving cell lysis, centrifugation, and supernatant containing protein.
Sample Prepartion Steps for Western Blot

The first step in sample preparation, is isolating the protein from a source. Usually, protein is purified from cells. However, other sources of protein, such as synthetically derived protein, can also be run through a Western blot.

Western blot sample preparation diagram showing addition of loading buffer to cell suspension, changing suspension color from light blue to dark blue.

Next, the protein concentration is determined. Loading buffer, which contains SDS (sodium dodecyl sulfate), and BME (beta-mercaptoethanol) is added to the protein suspension.

Western blot sample preparation diagram depicting denaturation and switch from a folded, native protein to a linear, unfolded protein.

The sample is then heated to near-boiling, which denatures the protein and allows the SDS to bind to the protein. SDS carries a negative charge.


Western blot SDS-PAGE schematic adding green protein-SDS sample solution to top of wells of gray gel.

The first step in SDS-PAGE, is placing the sample, containing the protein-SDS compound, in a well on top of the gel. A ladder is usually loaded in one of the wells. Ladders are protein mixtures of known molecular weight, that allow the researcher to determine the molecular weights of the other proteins on the gel.

Western blot SDS-PAGE showing protein separation in gel from the top negative pole of the gel to the bottom positive pole with proteins bands of green, red, yellow, and grey.

Once the sample(s) and ladder are loaded, a current is run across the gel, with a negative pole on the well end of the gel, and a positive pole on the opposite end of the gel. Because the protein is bound to negatively charged SDS, it is pulled down through the gel to the positive pole. The larger the protein, the slower it moves

Although running the gel is the last step in the SDS-PAGE method, it is important to make note of several pieces of information:

  • Proteins are separated by their size as they run through the gel.
  • The lower the concentration of acrylamide in a gel, the easier it becomes for proteins to move through the gel; so all proteins move further under the same conditions.
  • Gradient gels are gels where the concentration of acrylamide increases from the top to the bottom

Transferring Method

After the gel is run, it is placed against a membrane, and current is passed across the gel to the membrane, transferring the proteins onto the membrane.

Western blot transferring of proteins from grey gel to white membrane when a current is applied.

The transferring method detailed in this How To Guide is known as semi-dry, however transferring can also be done using the wet method. The membrane is usually made of PVDF or nitrocellulose, both of which have advantages and disadvantages that should be throughougly researched prior to use.

Immunoblotting Method

The first step in immunoblotting is to wash the membrane and block it with non-specific protein. The non-specific protein binds to the surface of the membrane where protein is not already present. This will prevent antibody from binding to the membrane and giving a non-specific signal.

First step of western blot immunoblotting involving blocking solution addition depicting be white membrane with colored protein bands changed to dark blue membrane.

Next, the primary antibody is added to the solution in which the membrane is floating. Remember that the primary antibody recognizes a specific amino-acid sequence of a particular protein.

Western blot primary antibody addition shown as light blue antibody added to dark blue membrane with antibody binding to green colored protein bands.

After a wash is conducted to remove unbound primary antibody, secondary antibody is added. Secondary antibody recognizes the primary antibody, and usually is conjugated with an enzyme, such as HRP (Horse Radish Peroxidase).

Western blot secondary antibody addition shown as green HRP molecule conjugated to dark blue secondary which detects the light blue primary antibody and is added to the dark blue membrane.

Lastly, another wash is performed to remove unbound secondary antibody. Non-specific binding of both the primary and secondary antibodies can occur, but thorough washing usually minimizes this problem. The amount of time the primary and secondary antibodies are applied, directly affects the specificity and strength of binding.

    Detection Method

    The detection method used is dependent upon the enzyme to which the secondary antibody is conjugated. The most common enzyme used in Western Blotting is HRP, and the substrate used for detection is known as chemiluminescent substrate. Bio-Techne's detection substrate is known as NovaLume, and NovaLume-Plus.

    Western blot detection showing that blue luminol substrate is oxidized by the green HRP enzyme conjugated to the antibody complex, producing a yellow light reaction.

    Once the substrate has been added, the light being emitted can be detected with film or a photo imager.

    Western blot detection showing substrate added to dark blue membrane and then exposure to grey film resulting in dark gray protein band detection.

    Notice that the film does not contain information on where the lit bands are located in relation to the membrane. Therefore, it is very important to use a method that allows the film to be aligned with the membrane in the exact same manner as when the film was exposed. The membrane is usually stained after the detection step, so that the protein present can be visualized, and compared to the film.

    Simple Western: Gel-Free, Blot-Free, Hands-Free

    Simple Western™ from ProteinSimple, a Bio-Techne brand, automates the Western blotting workflow using capillary electrophoresis to separate protein by size (CE-SDS) or charge (icIEF) followed seamlessly by immunodetection using traditional Western blot antibodies.