Western blotting is a technique used in life sciences and molecular biology labs to detect a specific protein in a variety of biological samples, such as blood or tissue.
The method is widely used in medical laboratories to diagnose diseases. In addition to detecting proteins, this technique is also used to visualize, differentiate, and quantify proteins in complex protein mixtures.
The Western blotting method involves separating proteins through gel electrophoresis methods (such as SDS-PAGE) based on their molecular weight (in kDa or kilo dalton) or size. Then, the separated proteins are blotted on nitrocellulose or PVDF membranes.
The membrane-containing protein is subjected to a primary antibody (often tagged with biotin proteins) specific to the target protein followed by an enzyme-conjugated (such as streptavidin-conjugate) secondary antibody. Then, suitable detection methods are used to study the binding of antibodies to specific proteins using suitable detection reagents; these can include radioactive or chemical tags.
Some detection techniques used in the Western blotting workflow are immunodetection, mass spectrometry, and N-terminal sequencing. In immunodetection, a primary antibody and AP- or horseradish peroxidase (HRP)-conjugated secondary antibody is used for colorimetric or chemiluminescent detection. As an alternative, fluorescently labeled primary or secondary antibodies can also be used to visualize the reaction products directly.
Figure: A Western blot protocol overview.
In this article, we explain the types of buffers used in the process, Western blot kits available in the market to perform Western blotting, and factors to consider when choosing the best kit for your workflow.
Previously, scientists needed to prepare their own buffer and reagents to carry out the blotting assay. However, with the advancement of technologies and fast pacing research and development in the industries, Western blot assay kits are now widely created and produced by manufacturers with the aim of shortening and simplifying the Western blotting process.
Some examples of Western blot kits available in the market for research use include a sensitive chemiluminescent kit, a Western blot detection kit, S-Nitrosylation Western Blot Kit and HENS Buffer, and Fast Western Blot Kit with ECL Substrate.
The components of the kit include:
By using Western blot kits and buffers, we can perform Western blotting in a fraction of the time it takes to prepare the workflow from scratch, starting with buffer preparation and reagent preparation all the way through detection and data analysis.
Western blot kits are whole packages of all the reagents, buffers, and antibodies you need to perform the Western blotting assay in their labs. Therefore, based on your goal and requirement you can incorporate them into your procedures.
You can either purchase a kit containing everything you need for end-to-end blotting assay or one that is only stage-specific. For example, there are some Western blotting detection kits available in the market to aid in the detection stage of the method. You can purchase them based on the detection method you want to use in your workflow.
Often these kits are sufficient to run 5-10 mini-gels (to find the exact number go through the datasheet provided by the distributor or check on their website using the catalog number of the product). These kits not only reduce the time involved in performing the method but also reduce the possible errors that might occur during reagent preparation.
Western Blot involves the use of a range of buffers, which include:
With the range of buffers and kits available in the market, it’s essential to ensure you get the right one for your lab. To help you out with the confusion, here are some factors that you should consider before putting your money into these Western blot kits.
Before buying a kit, it’s crucial to understand what you want to achieve with the workflow — what’s your goal?
To answer this question, ask yourself:
These answers will help you to invest in the right Western blotting kit.
Read carefully what the kit contains. Some kits have everything for protein detection ranging from buffers, antibodies (Goat Anti-Rabbit IgG or Goat anti-Mouse IgG Antibody/HRP-conjugate), protein ladders, chemiluminescent substrate (ECL), etc. while some contain only half of the reagents.
This is obvious but definitely check for reviews before purchasing from any supplier. You can do this online or talk to your colleagues to learn which companies’ or suppliers’ Western blot kits have been working for them.
Do take some suggestions from the old players in the field. Not only does it give you some confidence in making your choice but it also reduces the risk of buying a kit that produces false data or does not work up to your expectation.
Whatever kit you buy, ensure it contains a high-quality and accurate concentration of reagents to help you save some extra time and money and obtain reliable data.
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Western blot kits and buffers are optimized reagents offered by suppliers to cut short the time involved in performing Western blot assays — 4 hours of assay can come down to just 55 minutes. Additionally, they also help in reducing hands-on time and yield satisfactory, accurate, and measurable results.
With the range of Western blot and ELISA kits available in the market, you need to understand your goal, the detection methods you want to employ, and the content they contain, to invest in the right kit for your lab. Additionally, do ensure they are high-quality reagents from trusted suppliers.
This should be combined with high-throughput equipment for a smooth assay experience and to obtain accurate and reliable results. However, it often goes over the budget for small and individual labs. Even for big labs that do not perform these assays routinely, buying such expensive equipment for a short-needed-time can be a waste of money.
You can avoid this by using Excedr’s leasing program.
The program allows all sizes of labs (small or industrial size) to acquire their desired equipment on lease. There are no upfront costs involved or any additional repair and maintenance costs. We cover everything under the umbrella of your leasing program.
Further, there’s no limit on the number of equipment you can lease; get one or outfit your whole lab, the choice is yours!
With Excedr’s leasing solution, you can not save money but also gain the liberty to invest in your lab as per your preferences. By doing this, you can dedicate more time to your research, obtain vital accreditations, and achieve your objectives faster.