Western Blot Technique: Principle, Procedures and 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 mixture of proteins extracted from cells as well as evaluation of their size and amount. The SDS PAGE technique is prerequisite for western blotting.

Principle

Western blotting (protein blotting or immunoblotting) is a rapid and sensitive assay for 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 specific primary antibody and secondary enzyme labeled antibody and substrate.

Procedure

Western blotting procedures includes 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 localization of the protein of interest, solubilization of membrane bound proteins require 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 pipette. The cell mixture is centrifuged and 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

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 upon 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, 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 with running buffer that is appropriate for your gel chemistry.Rinse the wells of the gel with running buffer and add 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 principal 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  traditional wet transfer method are as follows:

Western Blotting Technique Test Procedure

Immunobloting:

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

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:

Detection can be done by other  methods such as:

Western Blot Test for HIV diagnosis

You can watch the following video to get more idea…

 

Uses

References and further readings: