Western Blotting Techniques and Processes

When electrophoretically-separated proteins are transferred or “blotted” from a gel matrix to a membrane for antibody-based surface detection this type of immunoblot is referred to as western blotting. This allows testing and analysis for specific target proteins in a complex mixture. It uses very selective, sensitive interactions between antigens and antibodies, providing data for both semi-quantitative and qualitative analysis.

Western Blot Analytical Technique

In western blotting, gel electrophoresis is used to separate natural proteins based on their 3D structure or denatured proteins by polypeptide length. These separated proteins can then b e moved onto a membrane, usually PVDF or nitrocellulose, where antibodies specific to the targeted protein stain them. It’s a commonly used technique in a range of molecular sciences, including immune genetics, biochemistry, molecular biology, and similar specialties within molecular biology. As these proteins are transformed into macromolecules, they are transferred or blotted onto immobilizing membranes to help with diagnosis, analysis, and detection of particular specific molecules.

During western blotting, proteins in the sample are separated by gel electrophoresis based on their isoelectric point, electric charge, molecular weight or a combination of these aspects using SDS-PAGE, IEF, and related methods. SDS-PAGE is commonly used for separation as all the proteins are soluble and will move in the same direction, making epitomes easier to access as the SDS denatures the molecules.

Western Blot Process

Once electrophoresis analysis is complete, proteins are moved from the gel onto a membrane so that they are available for antibody detection. PVDF membranes have a higher capacity to bind proteins, while nitrocellulose does a better job of binding small proteins. Electroblotting is the main method for transferring these proteins, using an electrical current to move the proteins from the gel onto the membrane. This can happen through tank blotting, semi-wet blotting, and semi-dry blotting. All of these methods create a sandwich of the gel and membrane with filter papers stacked on both sides. When isoelectric focusing is incorporated to separate the proteins, the proteins transfer more efficiently by diffusion. Electroblotting moves the proteins from inside the gel to the membrane while maintaining the order seen in the gel.

Taking about an hour to overnight, blotting is followed by exposure of the proteins on a thin layer of surface material to promote antibody detection. Because the membrane’s free binding sites are blocked by the protein mixture, it does not interfere with antibody probing following this process. Proteins are then detected based on the primary antibody, followed by a secondary antibody, that conjugates with specific molecules and is easily detected during the development process, which latches onto the primary antibody. This provides a high level of sensitivity in the testing process.

For the highest level of detection sensitivity, enhanced chemiluminescence is used, where the antibody recognized the primary antibody, a reaction between the substrate is matched to a secondary antibody that causes a chemiluminescent light to emit for a particular amount of time. The light is collected by exposing the membrane over an X-ray film or in a completely dark cabinet with recording by sensitive CCD cameras. Special variations of this process can detect protein bands down to 1 pg.

The western blot can also be used following protein phosphorylation, where an antibody binds to all isoforms of poly-phosphorylated proteins provides the appearance of beads on a string in 2D gel. Used for identifying known and unknown proteins in solutions brought through co-immunoprecipitation, this technique allows corresponding spots to be cut from duplicate gels and identified using related technology. This technique provides solid detection results to high levels of sensitivity and accuracy.

What is your reaction?

In Love
Not Sure

You may also like

Comments are closed.

More in:Health