Western blot technique | rocky ground (2023)

Western Blot (WB) or Immunoblot is a laborious immunoassay for most labs, used to demonstrate antibody specificity, confirm gene expression, detect post-translational modifications (PTMs), diagnose diseases, and much more. Specific detection of bands corresponding to the protein of interest results from successive probing of its blot with primary and secondary conjugated antibodies. It is a widely used method to detect a specific protein in a complex matrix such as a B. cell or tissue lysate (ie protein extracts).

The Western blot assay uses gel electrophoresis (SDS-PAGE or native PAGE) to separate proteins based on their molecular weight. Proteins are then transferred from the gel to a membrane (typically nitrocellulose or PVDF), which is then blocked with protein blocking buffer to prevent unspecific and probed binding.primary antibodyto detect the protein of interest. This is followed by incubation with asecondary antibodyconjugated to a reporter molecule, allowing visualization of the target protein; However, reporter-conjugated primary antibodies do not require the use of secondary antibodies. Washing steps with a buffer containing a mild detergent are also usually performed after antibody incubations to remove any non-specific binding.

Western blot experiments can be performed in a variety of formats, most of which require a secondary antibody conjugated as a reporter molecule. Reporter molecules include horseradish peroxidase, alkaline phosphatase enzymes and fluorophores. When using indicator enzymes, chromogenic or luminescent substrates can be used for detection.

Fluorophore reporter molecules do not require a substrate, but do require specialized equipment for data acquisition. Fluorescence detection is useful for multiplex WB experiments where multiple targets can be detected in the same assay using fluorophore conjugates with non-overlapping emission spectra. WB fluorescence is also ideal for quantitative analysis as detection allows for wide dynamic ranges and signal normalization.

The choice of WB membrane depends on the type of experiment to be carried out. The most commonly used membranes are nitrocellulose or polyvinyl difluoride (PVDF). Nitrocellulose is easy to use and provides adequate data for most common enzyme indicator experiments. Low fluorescence PVDF membranes are recommended for fluorescent Western blot applications.

Western blot technique | rocky ground (1)

Figure:Western Blot Procedure. 1. Cells expressing the protein of interest are harvested; 2. Cells are lysed and centrifuged to produce a protein suspension; 3. Proteins are separated by molecular weight using SDS-PAGE; 4. Proteins are transferred to a membrane by electroblotting; 5. The membrane is incubated with a primary antibody specific for the protein of interest followed by a secondary antibody conjugated to a reporter molecule; 6. The reporter molecule is visualized and photographed. (Created withBioRender.com)

(Video) Western Blot Method - Animated Video

WB multilisado

As specific guidelines and application standards for validating research antibodies become increasingly subject to scrutiny in the scientific community, a number of approaches can and should be implemented to demonstrate the specificity of an antibody of appropriate strength. One such approach is to confirm the properties of target-specific antibodies, includingcell lysatesÖcell linesthey are known to contain or not contain the target of interest based on genomic and proteomic studies. Appropriate negative controls for cells that naturally produce the target are the same cells in which target abundance is selectively altered by chemical stimulation or genetic approaches.

Rockland routinely tests the specificity of its primary antibodies by evaluating their performance on Western blots of various lysates. A variety of conditions are evaluated for each target so that specificity, sensitivity and reproducibility can be determined. This ensures reliable performance and confirms batch-to-batch consistency.

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Immunoprecipitation (IP) is one of the most widely used approaches for antigen detection and purification. The approach uses antigen-specific antibodies to isolate an antigen of interest from a complex mixture of proteins, which is then analyzed by Western blotting to determine the relative abundance and size of the target antigen itself and/or target-associated proteins.

Analysis of immunoprecipitated proteins by immunoblotting can be difficult because the reagent used to detect the WB staining antibody often binds to the heavy and light chains of the precipitating antibodies. As detailed below, this issue can be fixed easily with ourTrueBlot® Productsthat selectively bind to staining antibodies only through greater sensitivity, less background noise, and greater accuracy.

Western IP blots provide highly specific results, but often suffer from heavy/light chain transport, contamination and constant interference. TrueBlot® products solve almost all of these problems with greater sensitivity, less background noise and greater accuracy. With TrueBlot® reagents, you can generate clear, high-quality data in your immunoprecipitation and western blot protocols. Available in several options, fromTrueBlot® IP-Perlen,TrueBlot® Secondary Antibodiesto complete the IP/Western blot kits ofGoat,mausoleum, Örabbits.

west in the cell

Western Cell Assays (ICW) or cell-based assays, cell-based ELISA, cell-based ELISA (ICE) or rapid activation cell-based ELISA (FACE) provide researchers with a simple and efficient assay method for the rapid quantification of biomarkers . and signaling proteins in whole cells using antibodies. The LICOR® Odyssey® infrared imaging system is suitable for ICW; however, other imaging systems can also be used. The method combines the specificity of a WB with the quantification of an ELISA. ICW assays can be used for the detection of proteins in fixed cells or proteins with a physiologically or biologically relevant cellular context and in two-target multiplex quantitation using 700 nm and 800 nm channelsdye-conjugated antibodies.

Many of the Rockland antibodies have been validated for use in ICW, and since ICW assays are related to immunofluorescence (IF), most validated and approved antibodies for IF can also be similarly used in ICW studies.

Western blot technique | rocky ground (3)

Figure:The Western-in-cell technique allows multiplex antibody-based quantitation of two target proteins using 700 nm and 800 nm channels in fixed cells. (Created withBioRender.com)

(Video) Western Blot Protocol Overview

Secondary antibodies against WB

Western blot technique | rocky ground (4)

Secondary antibody conjugatesthey are ideal for western blot. When selecting a secondary antibody conjugate for an antibody assay, consideration should be given to the target species, conjugate (ie, peroxidase, FITC, biotin), and host species. In addition to standard secondary antibodies, Rockland offerspreadsorbed secondary antibodiessuitable for detection methods where cross-reactivity can be an issue.

Reporter enzymes are widely used in molecular biology because they allow the visualization or detection of immune complexes. Horseradish peroxidase (HRP) is a widely used reporter enzyme and can produce either a chromogenic or a luminescent (chemiluminescent) product, depending on the substrate. Alkaline phosphatase is also used, most typically as a reporter in the chromogenic Western blot assay format.

Alkaline phosphatase-conjugated antibodies (AP or Alk Phos) are used to detect proteins in ELISA immunoassays and Western blots. Alkaline phosphatase (AP) catalyzes colorimetric reactions withBCIP/NBT SubstrateÖFemtoMax Chemiluminescent Substrate. Secondary antibody conjugates are conjugated to the highest quality alkaline phosphatase using Rockland's proprietary technology.

Rockland conjugates a broad panel of secondary antibodies to many of the classic and state-of-the-art fluorescent markers, including fluorescein, Texas red and phycoerythrin. Rockland also produces many state-of-the-art fluorochrome dyes. These are designed for the detection of primary antibodies in multiplex multicolor assays. State-of-the-art fluorochrome conjugates (Atto-tec dyes, DyLight™ dyes) offer excellent absorption (high extinction coefficient), high fluorescence quantum yield and excellent photostability. All conjugates are ideal for various immunofluorescence-based assays, including fluorescence western blotting, immunofluorescence microscopy, FLISA, and more.

(Video) western blotting - the bumbling biochemist's guide

Substrates for WB

Chromogen Substrate

Chromogenic substrates are used in colorimetric assays because they result in a measurable color change in the presence of an enzyme-antibody complex bound to specific analytes. For WB with horseradish peroxidase (HRP) in colorimetric detectionTMBjDAB-Substrateare commonly used. Alkaline phosphatase (AP) chromogenic substrates includeBCIP/NBT, which generally have the highest sensitivity and reliable detection of AP activity. Rockland Chromogenic Blotting substrates are available in a variety of specifications and formats. The appropriate choice of substrate depends on the enzyme label, the desired sensitivity, and the signal form or detection method required.

The peroxidase reaction with ourTMBM substrateproduces a blue, water-soluble product that can be precipitated on a membrane. The precipitated product produces blue to dark blue bands at the enzyme site. The TMBM is suitable for applications that require a high signal-to-noise ratio. DAB is another peroxidase substrate and produces a brown precipitate in the presence of HRP and peroxide.

The NBT/BCIP reagent is also commonly used in chromogenic Western blot immunoassays. NBT serves as an oxidizing agent and BCIP as a substrate for alkaline phosphatase. Together, NBT and BCIP, in the presence of alkaline phosphatase, form reagents that produce a dark purple to black water-insoluble precipitation product that offers strong sensitivity.

Chemiluminescent substrates

Western blot technique | rocky ground (5)

Rockland manufactures several luminol-based chemiluminescent substrates for the detection of horseradish peroxidase (HRP). HeFemtoMax™ Supersensitive HRP SubstrateIt is designed for high performance Western blotting and works on nitrocellulose and PVDF membranes. FemtoMax™ generates chemiluminescence and allows the detection of amounts of antigens up to femtograms (10-15). Detection methods may include photographic film or other imaging methods, including systems based on highly sensitive CCD cameras.

Chemiluminescent substrates offer several advantages over chromogenic substrates. In principle, these systems are much more sensitive to detect enzymatic activity without the use of radioactive isotopes, luminescence detection usually occurs within a few minutes and the signal is better quantified as it requires the use of charge-coupled digital devices (CCDs) for detection, which allow a large dynamic range with long exposures.

Lock Buffer for WB

When performing a western blot, the block buffer must not be ignored. The blocking buffer fills in the membrane sites that can bind proteins and cause background imaging if left untreated. The critical reagent in a blocking buffer is the protein, which protein does not react with antibodies. Popular blocking proteins include skim milk powder (NFDM), BSA, casein and combinations thereof. It should be noted that a single blocking agent may not be sufficient for all Western applications. Some blockers may interfere with primary antibody activity, be incompatible with the reporter system used, or cause unwanted autofluorescence. We have developed several blocking buffer reagents suitable for all Western blot applications, includingBLOTTO-NFDMjBSAfor standard applications and a special recipeFluorescence Western Blot Blocking Buffer.

Fluorescence Blocking Buffers are specially formulated to preserve the fluorescence of secondary antibodies conjugated to fluorochrome dyes and also provide extremely low background compared to other blocking buffers. Our buffers work with many fluorescent secondary antibody conjugates including FITC, IRDye®, Atto Dye and DyLight Dye. Our Fluorescence Western Blot Blocking Buffer is excellent for standard chemiluminescence blotting applications and has been shown to be superior for 2D WB experiments when directly compared to other blocking reagents.

(Video) Lecture 53 : Western Blotting Technique

Kits para WB

Western blot technique | rocky ground (6)

Kits de Western-Blotmay be available for your assay, simplifying your reagent needs. We offer kits for chemiluminescence, fluorescence and chromogenic immunoassay formats. Our kits come with simple protocols that are easy to use for novice and experienced users alike. Kits are species specific for the detection ofmausoleumÖrabbitsPrimary and Antibodies are supplied as a format-specific package containing a Membrane Blocking Reagent, Wash Buffer, Secondary Antibody, and Substrate (if needed). Some of our most popular kits include FentoMax™ kits for chemiluminescent applications (human,mausoleum,rabbits,Goat) and Dylight™ kits for detection in fluorescent Western blot protocol imaging systems.

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Which 3 techniques does the western blot employ? ›

The technique uses three elements to accomplish this task: (1) separation by size, (2) transfer to a solid support, and (3) marking target protein using a proper primary and secondary antibody to visualize.

What are the 5 steps of western blotting? ›

Five steps are involved in western blotting procedure and detection assay, namely, transfer, blocking, primary antibody incubation, secondary antibody incubation and protein detection, and western blotting analysis.

What is the technique of western blotting? ›

Western blotting is a laboratory technique used to detect a specific protein in a blood or tissue sample. The method involves using gel electrophoresis to separate the sample's proteins. The separated proteins are transferred out of the gel to the surface of a membrane.

What are the 6 steps of western blotting? ›

There are six steps involved in western blot, including sample preparation, gel electrophoresis, proteins transfer, blocking, antibody incubation, and proteins detection and visualization.

What is the difference between the three blotting techniques? ›

Different blotting is used to detect different type of macromolecules such as southern blotting is used for DNA analysis, western blotting is for protein analysis, northern blotting is for RNA analysis and eastern for carbohydrate detection.

What is the most common detection method in western blot? ›

Enzymatic detection. One of the most common methods of detection in western blot is based on the use of a secondary antibody conjugated with an enzyme. The secondary antibody provides binding specificity towards the target protein recognized by the primary antibody, while the attached enzyme is used for detection.


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