Western Blot Technique: Principle, Steps, Uses

The term “blotting” refers to the transfer of biological samples from a gel to a membrane and their subsequent detection on the surface of the membrane. Southern blot is used for transferring DNA, Northern blot for RNA, and Western blot for Protein. Western blotting (also called immunoblotting, because an antibody is used to specifically detect its antigen) was introduced by Towbin, et al. in 1979 and is now a routine technique for protein analysis.

Western blotting can produce qualitative and semi-quantitative data about the protein of interest. It is an important technique used in cell and molecular biology. It enables the researchers to identify the specific protein from a mixture of proteins extracted from cells as well as evaluation of their size and amount. The SDS PAGE technique is a prerequisite for western blotting.

Principle

Western blotting (protein blotting or immunoblotting) is a rapid and sensitive assay for the detection and characterization of proteins. It is based on the principle of immunochromatography where proteins are separated into polyacrylamide gel according to their molecular weight.

The protein thus separated are then transferred or electrotransferred onto nitrocellulose membrane and are detected using a specific primary antibody and secondary enzyme-labeled antibody and substrate.

## Procedure

Western blotting procedures include the following steps:

Tissue Preparation (preparation of sample lysate)

Take the sample, add ice-cold PBS and lysis buffer such as RIPA buffer which is a commonly used buffer for maximum protein yield.  (The choice of lysis buffer largely depends on the localization of the protein of interest, solubilization of membrane-bound proteins requires stronger extraction detergents compared with isolated cytoplasmic proteins).

Always use freshly prepared protease inhibitors, keep samples on ice and work quickly.

Lysis buffer should contain protease inhibitors to prevent the degradation of the protein of interest.  Cells are lysed by incubating on ice and later applying shear pressure using a pipette. The cell mixture is centrifuged and the pellet is discarded. The supernatant is the lysate which we will use for further processing.

Western blots are typically performed under reduced and denatured conditions. These conditions will allow proteins to be separated by their molecular weight rather than their native conformational shape or charge. To reduce and denature samples dilute each in a loading buffer such as Laemmli sample buffer. This buffer contains

• beta-mercaptoethanol, or DTT, to reduce disulfide bridges between cysteines,
• SDS to assist in denaturing and to provide a net negative charge to the protein,
• glycerol to allow the samples to sink into each well,
• bromophenol blue to visualize the lysate and an ionic buffer.

Vortex each sample and incubate at 95 degrees Celsius for five minutes to completely denature the proteins.  Now the sample is ready to load into an SDS page gel.

Gel Electrophoresis

In this step, we will separate the individual proteins in our sample lysate based on their molecular weight using a positive electrode to attract a negatively charged protein. To do this, we load our previously prepared protein samples into a commercially available polyacrylamide gel.

Gels are available in fixed percentages or gradients of acrylamide. The higher the acrylamide percentage the smaller the pore size of the gel matrix. Therefore higher percentage of gels are better for low molecular weight proteins, a low percentage of gel are useful for large proteins and gradient gels can be used for proteins of all sizes due to their varying range in pore size.

1. Prepare your gel by inserting it into the electrophoresis apparatus and filling it with a running buffer that is appropriate for your gel chemistry. Rinse the wells of the gel with a running buffer and add a buffer to the chambers.
2. Load your samples into the wells and load a  pre-stained molecular weight ladder into one well.  The ladder will allow you to monitor protein separation during electrophoresis and subsequently verify protein weight in your sample during later analysis.
3. Close the electrophoresis unit and connect it to a power supply. Most units typically run 45-60 minutes at 200 volts or until the loading buffer reaches the bottom of the gel. During this time the negatively charged proteins in each sample will migrate toward the positively charged electrode making their way through the polyacrylamide gel matrix.

Transfer

In this next step, we will transfer separated proteins out of the gel into a solid membrane or blot. This is based upon the same principle as the previous step in which an electric field is charged to move the negative proteins towards a positive electrode. Transfer can occur under wet or semi-dry conditions.

The steps of the traditional wet transfer method are as follows:

• Start by removing the gel from its cassette and cutting the top portion containing the wells.

• Notch the top left corner to indicate gel orientation.

• Float the gel in transfer buffer while preparing the transfer sandwich. To make the transfer sandwich,  a cassette, sponges, filter paper, gel, and  PVDF or nitrocellulose membrane paper is needed.

• Notch the top left corner of blotting paper to indicate blot orientation and incubate membranes in transfer buffer for 10 minutes.

  

• Create a stack by placing the following components from the black negative cathode to the red positive anode: sponge, filter paper, gel, membrane, filter paper, and sponge (Be careful not to touch the gel or membrane with your bare hands and use clean tweezers or spatula instead. Touching the membrane during any phase can contaminate the blot and lead to an excessive background signal).

• Use a clean roller with each layer to gently roll out any bubbles that may be present since bubbles will inhibit efficient protein transfer.

• Lock the cassette and place it in the transfer apparatus containing a cold transfer buffer ensuring that the cassette is properly positioned from negative to positive. In order to prevent heat buildup, it is beneficial to transfer with a cold pack in the apparatus or in a cold room with the spinner bar placed at the bottom of the chamber.

• Close the chamber and connect to a power supply.

• Perform the transfer according to the manufacturer’s instruction which is normally 100 volts for a third to 120 minutes.

Figure: Western Blotting Technique Test Procedure

Immunoblotting

After electrotransfer of protein to a membrane, we will now block the blot by applying a primary antibody specific for our protein of interest and then a secondary antibody that will recognize the primary antibody. Steps:

• Start by removing the membrane from the cassette and rinsing it three times in water.
• As an optional step, we can verify the proteins were transferred successfully by staining the membrane with ponceau red. Incubate the membrane in ponceau for five minutes and wash with water until the bands are clear. After verification the blot can then be de-stained by continuing to wash with water or TBS tween until the dye is completely removed.
• We need to block all areas of the blot which do not already contain protein. This will prevent the non-specific binding of the antibody and reduce the overall background signal.
• Common blocking buffers include 5% non-fat dry milk or BSA in a TBS-Tween solution. However, do not use a milk solution when probing with phosphor-specific antibodies as it can cause high background from its endogenous phosphoprotein, casein.
• Incubate the membrane with blocking solution for one hour at room temperature under slight agitation.
• Decant the blocking solution and wash with TBS tween for five minutes.
• We are not ready to add our primary antibody. Dilute the primary antibody in a blocking buffer at the concentration recommended on the datasheet and incubate overnight at 4 degrees Celsius with gentle shaking.
• A recommended optional step is to also use a positive loading control antibody which allows the user to verify equal amounts of total protein were loaded into each well and aids in troubleshooting by removing any uncertainties with the Western Blot procedure.
• Next day: decant off the primary antibody and wash the membrane with large volumes or TBS tween and vigorous agitation five times for five minutes each. These stringent washes are extremely important for removing non-specific background signals.
• After washing, dilute the secondary antibody in the blocking solution and incubate the membrane for one hour at room temperature at the concentration recommended on the datasheet. In our example, the secondary antibody is also conjugated to HRP for later detection.
• Decant membrane and wash the membrane with large volumes of TBS tween with vigorous agitation five times for five minutes each. You are now ready for the detection phase.

Detection

In this final phase, we will demonstrate signal development using the most common, most sensitive, and most inexpensive detection method the electrochemiluminescence or ECL reaction. This method utilizes the HRP enzyme which was conjugated to the secondary to catalyze the ECL reaction and produce light. A light is then gathered onto x-ray film and developed or digitized with the aid of a specialized camera sensitive enough for this application.

### Steps

• We start by mixing equal parts ECL reagents in a one-to-one ratio according to the manufacturer’s instructions.
• We will incubate the membrane for 3-5 minutes without agitation.
• After incubation, decant ECL mixture and use a laboratory wipe to wipe off the excess solution from the corner of the membrane.
• Place the membrane in a clear plastic wrap such as a sheet protector to prevent drying.
• We can now use a roller to push out any bubbles or any excess solution.
• Immediately develop the membrane.
• Both film and camera systems allow us to manually adjust the exposure time in order to ensure a picture-perfect Western Blot. Relative band densities can now be quantified with commercially available software. Proper molecular weight can also be verified by comparing band sizes to the molecular weight ladder.

Detection can be done by other methods such as

Colorimetric detection

It depends on the incubation of the western blot with a substrate that reacts with the reporter enzyme (such as peroxidase) that is bound to the secondary antibody. This converts the soluble dye into an insoluble form of a different color that precipitates next to the enzyme and thereby stains the membrane. Development of the blot is then stopped by washing away the soluble dye. Protein levels are evaluated through spectrophotometry.

Radioactive detection

Radioactive labels do not require enzyme substrates, but rather, allow the placement of medical X-ray film directly against the western blot, which develops as it is exposed to the label and creates dark regions which correspond to the protein bands of interest. The importance of the radioactive detection method is declining due to its hazardous radiation because it is very expensive, health and safety risks are high, and ECL (enhanced chemiluminescence) provides a useful alternative.

Fluorescent detection

The fluorescently labeled probe is excited by light and the emission of the excitation is then detected by a photosensor such as a CCD camera equipped with appropriate emission filters which captures a digital image of the western blot and allows further data analysis such as molecular weight analysis and quantitative western blot analysis. Fluorescence is considered to be one of the best methods for quantification but is less sensitive than chemiluminescence.

Figure: Western Blot Test forHIV diagnosis

You can watch the following video to get more idea

Uses

• It is the most sensitive and specific test for determining the size and amount of protein present in any material.
• The confirmatory HIV test employs a western blot to detect anti-HIV antibodies in a human serum sample.
• A western blot is also used as the definitive test for Creutzfeldt-Jakob Disease, Lyme disease,  hepatitis B infection, and HSV-2 (Herpes Type 2) infection.

References and further readings:

• NOVUS Biologicals. You can find detailed information regarding reagent preparation here: