Last Updated on
June 13, 2023
Western blotting is a technique used in research labs to detect specific proteins in a given sample. During the process, a mixture of proteins is separated through SDS-PAGE or sodium dodecyl-sulfate polyacrylamide gel electrophoresis, based on their molecular weight.
The obtained proteins are then transferred on the surface of a membrane, generally polyvinylidene difluoride (PVDF) or nitrocellulose membrane, through electrophoretic transfer. The blotting membrane containing protein is then incubated with antigen-specific antibodies. Often primary and secondary antibodies are used in the process.
The binding of the antibodies to the target protein is detected using several detection techniques, such as enzymatic, fluorescent, colorimetric, or chemiluminescent approaches. A typical substrate for detecting proteins is ECL, which is used for detecting horseradish peroxidase (HRP) on immunoblots.
Western blot assay has a myriad of applications in life sciences, biology, and diagnostic lab workflows for protein separation, sequencing, target protein detection, diagnosis of diseases, and many more.
Other than SDS-PAGE gels, native gels are also used in western blot protocols, especially when denaturation of protein is not needed. Both these gels slightly differ from each other, which you’ll definitely want to understand for the success of your research.
Further, the SDS-PAGE gels are also of two types: fixed and gradient gels. Both of these differ in the concentration of acrylamide used in their preparation, and both of them prefer a different range of protein molecule weight.
In this article, we will review the different types of equipment one requires to perform the quantitative western blot assay and understand how they help in your workflow.
The western blot equipment and reagents can be understood better by dividing the protocol into its different stages: Sample Preparation, Gel Electrophoresis, Protein Transfer, Antibody Incubation, and Protein Detection.
To perform these processes, one will need equipment like:
The above ones are just to name a few. But, the one thing to make sure of while acquiring any reagent or equipment for your workflow is the quality of the materials and chemicals. The reagents should be lab-grade and high-quality, whereas, the equipment should be advanced, high-throughput machines that can provide you with accurate and precise results.
Blotting requires a range of equipment from the stage of protein transfer from the gel to the membrane to membrane processing and imaging. For example, the blotting stage requires a blotter for wet, dry, or semi-dry conditions.
Gel electrophoresis instruments are used to run and compare proteins from different samples in forensic, microbiology, and molecular biology labs. They separate proteins based on their molecular weight and charges.
The two basic electrophoresis instruments used in the process are horizontal and vertical gels. In the horizontal instrument, the sample is placed on the negative side of the equipment and when electrodes are attached and the power supply is on, proteins start moving from the negative side to the positive side of the gel system. They are required to separate larger sample sizes. Similarly, in the vertical system, proteins are loaded at the top and move downwards power is given.
There are also other factors to consider when choosing a gel electrophoresis instrument, such as the number of wells and the capacity of the wells, as well as the availability of the timed and untimed programs.
An imaging system in a western blot allows the visualization of target proteins in a protein mixture. Today, a range of instruments are available that integrate western blot documentation and gel imaging. Each of these types of techniques and imaging instruments enables researchers to achieve better documentation and imaging of their protein of interest.
For visualization, the gel samples are normally stained using reagents like ethidium bromide (EtBr) or other similar fluorophores.
Protein levels are traditionally detected using an X-ray film or a transilluminator. X-ray film is used to detect target proteins in chemiluminescent western blots, which require a darkroom. However, modern detection methods involve a digital charge-coupled device (CCD) imager to detect desired molecules.
Western blots and gel doc systems with advanced software are essential for researchers to automatically acquire, analyze, and validate the data — especially when you work with a spectrum of protein staining modes, such as colorimetric, fluorescent, radioisotopic, and chemiluminescent, it ensures accurate sample preparation and practical data storage.
The filter papers used in western blotting assays are composed of high-quality alpha cotton cellulose fiber. They help the quick and efficient transfer of the protein bands from gels to nitrocellulose or PVDF membranes. To minimize background signal sources, they are activated with ultrapure water without additives. In protein blotting workflows, they can be used with alcohol or other organic solvents.
To transfer protein bands from gels to the membrane, a range of tank blotting transfer systems are used. These systems are available in mini, midi, and large designs for qualitative and quantitative transfer of proteins of different molecular weights. Some systems are designed to transfer multiple gels simultaneously in one go.
However, among all these systems the wet transfer cell is the easiest, most efficient, and most common western blot transfer approach because of its throughput.
Semi-dry protein transfers are conducted by placing the transfer sandwiched horizontally between two plate electrodes. It can transfer proteins of molecular weight ranging from 10 to 100 kD in 15 to 60 minutes, though today there are also some systems that can transfer proteins of up to 400 kD in just three minutes.
The semi-dry transfer system allows efficient, fast, and economical blotting in the absence of a buffer tank or gel cassettes.
Western blotting is a useful technique used in molecular biology and biotechnology labs to detect and study a range of known and unknown proteins from a protein mixture. The workflow requires high-quality reagents and high-throughput equipment to obtain accurate and precise data in research applications.
The five most common types of western blotting equipment used by researchers in labs include gels, imaging systems, filter papers, tank blotting transfer systems, and semi-dry blotting systems. The advanced and automated version of this equipment allows more efficient work, increases your lab productivity, and better data storage.
However, when you’re just building your lab or don’t have an adequate budget, it becomes difficult to invest in acquiring high-throughput equipment. That’s why Excedr makes it easier for you.
Excedr allows labs of all sizes to lease either one piece of equipment or outfit their whole lab with all the desired equipment they need. There are no expensive upfront payments or additional repair and maintenance costs. Excedr’s comprehensive leasing solution covers all under its umbrella. Further, the team is always on standby to help you with all your questions or assist you in operating the equipment.
Excedr’s leasing solution not only saves you the time and money that you put into buying each piece of equipment but also allows you to focus your energy on your research and spend on your lab operations as you want.