Western Blot Analysis

Detection of specific proteins through Western blot analysis has been an essential tool in biochemical laboratories for many years. This widespread use is owed to the straightforward technique that combines protein separation and antibody-based immunodetection. With appropriate controls, the Western blot may also be used semi-quantitatively, providing useful information on the level of expression of target genes. The application is also used in identifying post-translationally modified proteins in both healthy and disease states.¹ In this product catalog, we summarize the Western blot workflow while highlighting some defining tools and equipment.

The Western blot procedure

While Western blotting procudures can vary slightly depending on the experimental goals, the overall workflow can be broadly summarized as follows:


Protein Extraction: At the start of any immunoblotting experiment is the collection of protein samples. When preparing samples from cell cultures, primary cells, or tissues, the cells must be lysed to release proteins. This can be done via homogenization, chemical lysis, freeze-thaw and sonication. The resulting total protein lysate contains all of the proteins in the cell, including cytoplasmic, membrane and nuclear proteins. The extraction will often include protease inhibitors to prevent unintended protein degradation. An effective protein preparation process will help ensure that samples will accurately represent the cell population's phenotype. Finally, a protein quantification assay should be performed to determine the protein concentration of the sample.

Protein Electrophoresis: Protein gel electrophoresis separates proteins by size, generally under denaturing conditions. SDS-PAGE is often used, which uses a polyacrylamide gel matrix and buffers containing the detergent SDS. To prepare SDS-PAGE gels, a resolving portion is cast first, followed by the stacking portion, upon which protein samples will be loaded. Protein electrophoresis gels, which run under a discontinuous buffer, use vertical electrophoresis systems. This is in contrast to the horizontal systems used in DNA gels. Prior to loading, samples are prepared with the addition of a gel loading buffer containing dye, SDS, glycerol, and reducing agent (such as DTT, TCEP or ßME). Loading control samples are often included here to account for any loss of protein during subsequent steps, as well as a protein standard to determine protein size. Electrophoresis yields a gel slab of separated proteins ready to be transferred onto a blotting membrane.

Blot Transfer: The transfer of proteins from a polyacrylamide gel onto a blotting membrane can be carried out using a wet or semi-dry electrotransfer device. Wet blot systems require a gel and membrane to be sandwiched and immersed in buffer, followed by continuous electrophoresis. In contrast, semi-dry blotters use significantly less transfer buffer and operate much quicker. The two main choices for blotting membranes are PVDF and nitrocellulose. PVDF exhibits a greater binding capacity than nitrocellulose, making it ideal for proteins with lower abundance. Nitrocellulose , while not as sensitive, is known to benefit from lower background noise. After the transfer, the transfer efficiency can be asssessed by staining the membrane with a reversible protein stain, such as Ponceau S.

Immunostaining: Immunostaining, also known as immunoblotting, is the use of antibodies to bind and detect target proteins embedded in the blotting membrane. This stage begins with the initial blocking of the membrane with the addition of a blocking buffer. These are solutions of mixed proteins, such as serum or milk, that help ensure antibodies will bind only to their intended epitope. After washing, the membrane is incubated with primary antibodies that are specific to the the target protein(s) of interest. Another round of blocking and washing is followed with incubation with secondary antibodies. These are conjugated with enzyme labels, generally alkaline phosphatase or horseradish peroxidase, that facilitate the detection of the primary antibody. Detection enzymes require the addition of substrates, which produce colorimetric, fluorometric, or luminescent signals. Secondary antibodies conjugated with fluorescent labels or biotin are also available. The choices of primary and secondary antibodies offers a great deal of flexibility for the Western blot user due to the different varieties of host species and detection methods.

Blot Imaging: Producing a clean image of a blot with clear, defined protein bands and controls marks the successful conclusion of the Western blot procedure. This type of imaging is now often carried out by digital imaging systems, including gel documentation imagers and other imagers that capture chemiluminescence or fluorescence. These instruments enable the visualization of blots by illuminating the membrane, recognizing signals over background noise, and capturing the image. Specialized imagers also often come with analysis software that can measure relative band intensities, allowing for both qualitative and quantitative Western blot analysis.

Western blotting tips and best practices

Fresh beats old: When issues are observed in the results, an often easy fix is to replace older reagents with freshly prepared batches. For example, APS and TEMED, which are used in polymerizing polyacrylamide gels, quickly degrade over time. These should be used fresh and added last when the gels are ready to be cast. In addition, once both the stacking and resolving layers of the protein gel is cast, the electrophoresis should be performed soon. Users conducting many Western blot experiments who wish to save time may consider using precast gels, which offer a longer shelf life. Fresher protein samples are also more ideal. Older or frozen protein lysates are more subject to protein loss or degradation, which can be caused by sustained protease activity and spontaneous degradation. This can lead to more noise or missing bands, so it is advisable to use fresh lysates whenever possible.

Minimize misinterpretation: As a multi-step process, Western blotting has some risk of protein loss. The effective use of controls can help ensure that the observed immunoblotting results provide a true reflection of the physiological state. Users should consider including a loading control, a range of relevant primary antibody concentrations, a positive control lysate, and a negative lane omiting the primary antibody. ² Ponceau S staining, which should be used to determine the success of the blot electrotransfer, may also be used for total protein normalization. The use of total protein normalization over housekeeping loading controls has received wide support in recent years.¹ Total protein can be detected either by staining the protein gel before transfer or staining the membrane after transfer and before blocking. An image analysis software can then be used to calculate the amount of total protein in a lane to normalize the signal of the band of interest.³

Choose the antibodies carefully: Finally, it is crucial that the chosen antibodies used for immunoblotting are specific and produce reproducible results. The presence of multiple or incorrectly sized bands can often be attributed to poor antibody specificity. Ideal primary antibodies should be specific to the target of interest and should not cross-react with any other proteins. They should also be raised in a different species than the sample to minimize secondary antibody binding to sample immunoglobulins.⁴ Users should carefully examine the validation data provided by the supplier, including data figures and the experimental conditions used to obtain the results. It is also ideal that prior to experiments, researchers should perform their own validation and optimization tests on antibodies, including both positive and negative controls.¹ Optimization of the primary and secondary antibodies will also prevent oversaturation of the signal, allowing accurate quantification.³

Discover useful tools for Western blotting

Browse our catalog to discover many useful Western blotting tools from trusted suppliers ranging from reagent sets to automated instruments. When purchasing supplies, the type of sample to be analyzed should be taken into consideration. Requirements for a small, relatively pure sample will be very different than those for a crude lysate or other complex mixture. Determine also the intended throughput and scale of your team's experiments. Automation-compatible devices may be worthwile for high-volume projects.

References

1. Sule R, Rivera G, Gomes AV. Western blotting (immunoblotting): history, theory, uses, protocol and problems. Biotechniques. 2023;75(3):99-114. doi:10.2144/btn-2022-0034

2. Tanabe L. The Ultimate Quick Tips & Tricks Guide to Western Blots. Biocompare. 2024 May 9 [cited 2024 May]. Available from: https://www.biocompare.com/Editorial-Articles/612493-The-Ultimate-Quick-Tips-Tricks-Guide-to-Western-Blots/

3. Easthope E. How to Improve Western Blot Reproducibility. Biocompare. 2023 Jul 18 [cited 2024 May]. Available from: https://www.biocompare.com/Editorial-Articles/597513-How-to-Improve-Western-Blot-Reproducibility/

4. Easthope E. How to Produce Publication-Worthy Western Blots. Biocompare. 2023 Feb 7 [cited 2024 May]. Available from: https://www.biocompare.com/Bench-Tips/594218-How-to-Produce-Publication-Worthy-Western-Blots/