Western Blotting Assays

Western blotting is an analytical technique used to detect the presence of specific proteins in a complex biological sample. This process involves the electrophoretic separation of proteins, the transfer of separated proteins from a gel to a stable membrane substrate, and their subsequent detection by antibodies labeled with reporter molecules. Antibodies are selected based on their specificity for the protein of interest, and this specificity of the antibody-protein interaction enables target identification. Common reporter molecules include enzymes, such as horseradish peroxidase (HRP) and alkaline phosphatase (ALP), or fluorescent dyes, such as iFluor® 488, with the latter affording greater sensitivity and multiplexing capacity.

Western blotting is used extensively in biochemistry applications targeting complex proteins, testing for disease markers (e.g., HIV, Lyme disease, or Hepatitis B), and confirming protein production in cloning experiments. With the development of novel blotting technologies and specialized automated equipment, western blotting has become a cornerstone in proteomics research and protein detection, producing qualitative and semi-quantitative results.

Western Blotting Workflow

Electrophoretic Separation of Proteins

Three-fold dilution series of BSA standards were separated on a NuPAGE^® 4-12% Bis-Tris gel and stained with A) ProLite™ Orange Protein Gel Stain or B) Coomassie brilliant blue (CBB) according to standard protocols. The ProLite™ Orange stained gels were photographed using a SYPRO Orange filter. The CBB-stained gels were photographed using transmitted white light without an optical filter. Lane 1: 15µg, Lane 7: ~20ng, Lane 10: ~0.8 ng BSA.

Like many characterization techniques, western blotting uses gel electrophoresis to separate proteins based on size and charge. In this method, an electric field forces proteins through the pores of a gel. Smaller proteins migrate faster than larger ones due to less resistance from the gel until pockets within the gel encapsulating proteins of similar size and charge are produced. Gels typically have multiple channels or wells that separate several related experimental mixtures. For accurate size determination, a protein standard is run in conjunction with the sample. Standards include several proteins ranging in size from 10 to 250 kDa for comparison purposes and are available in various formats. The PageTell™ Prestained 10 to 250 kDa Protein Ladder (Cat No. 60080 and 60081) is a three-color protein standard that includes 13-prestained proteins ranging from 10 to 250 kDa in size. Eleven of the proteins are covalently coupled with a blue chromophore except for two reference bands, a green and a red band at 25 kDa and 75 kDa, respectively, and when separated form distinctly visible bands. PageTell™ protein standards can be conveniently loaded directly onto gels without any preparation (e.g., heat or additional loading buffer).

Since proteins are complex macromolecules folded in on themselves, western blotting often uses a procedure such as PAGE (polyacrylamide gel electrophoresis) to provide information about its mass, charge, purity, and presence. There are several forms of PAGE, such as non-denaturing PAGE or native PAGE, which separate proteins based on their mass-to-charge ratios, but the most common is SDS-PAGE. In SDS-PAGE, the detergent sodium dodecyl sulfate is used to denature proteins and normalize their mass-to-charge ratios. The subsequent application of heat is used to remove the secondary and tertiary protein structure points so that each protein presents as one long linear macromolecule. In addition, SDS applies a negative charge equally across the length of the protein so that each molecule's movement through the gel, as driven by the electric field, is determined by its molecular weight only. Once separated by electrophoresis, proteins can be visualized in a gel using various stains, such as ProLite™ Orange (Cat No. 18000), or transferred onto a membrane for western blot analysis.

Transferring Proteins to the Membrane

Once the mixture of proteins has been separated, the pattern is transferred onto a membrane for detection. Western blot membranes are either nitrocellulose (NC) or polyvinylidene difluoride (PVDF), which have a high affinity for proteins. Most often, the separated pockets of protein are transferred onto the membrane using electroblotting or electrophoretic transfer; this method pulls the negatively charged molecules through the cross-section of the gel and onto the membrane, where they bind to the surface. When transferred to the membrane, the separated pockets of molecular weight maintain their physical position.

Blocking nonspecific sites

After transfer, it is critical to block any remaining sites on the surface of the membrane to prevent the nonspecific binding of detection antibodies during subsequent steps. Various buffers, including skim milk, bovine serum albumin (BSA), and highly purified proteins, have been successfully used as blocking buffers in western blotting. Consequently, determining the appropriate blocking buffer is system-dependent. Since the efficacy of blocking agents can vary between applications, empirically testing various blocking buffers can help achieve optimal results. Nevertheless, the blocking buffer should improve the sensitivity of the assay by reducing background interference and improving the signal-to-noise ratio.

Detecting Proteins Using Primary and Secondary Antibodies

In the final steps of the process, the blocked membrane is probed with primary antibodies specific to the protein or epitope of interest. In most cases, primary antibodies are unlabeled, and tagged secondary antibodies are used to bind to the primary antibodies and assist in detecting the target antigen. This type of strategy, called indirect detection (figure 2), offers several advantages, including signal amplification, enhanced sensitivity, and flexibility. However, additional incubation and wash steps are required. The choice of secondary antibody will depend on either the host species of the primary antibody or any hapten or other tag (e.g., histidine (His-tag) or hemagglutinin (HA-tag)) bound to the primary antibody. For example, if the primary antibody is a mouse monoclonal, an anti-mouse secondary antibody is required from a non-mouse host. Once antibodies have been attached, the membrane is ready for review.

Representation of direct and indirect detection. Indirect detection affords the binding of multiple secondary antibodies to each primary antibody resulting in significant signal amplification (created with BioRender.com).

For multiplex western blot analysis, highly cross-adsorbed secondary antibodies are recommended to minimize cross-reactivity between antibodies. These antibodies are manufactured with an additional purification step to remove antibodies that bind to off-target immunoglobulin (IgG) species.

Western Blotting Detection Methods

Detection methods for visualizing a western blot can be colorimetric, fluorescent, chemiluminescent, or radioactive. Radioactive markers were used in the past but are no longer favored due to the expense and special handling required. Nowadays, secondary antibodies tagged with fluorophores or enzymes (e.g., HRP) are used to detect proteins of interest.

Enzymatic Labels

Horseradish peroxidase (HRP) and alkaline phosphatase (AP) are the two enzymes most commonly used for protein detection. Enzyme-conjugates offer the most flexibility in detection as an array of chromogenic, fluorogenic, and chemiluminescent substrates are available for use with either enzyme. Of the three, chromogenic substrates are the simplest to use, cost-effective, and provide direct visualization of signal development (i.e., enzyme-substrate reaction results in colored precipitate). Chemiluminescent substrates differ from other substrates in that the enzyme-substrate reaction produces light as a byproduct. In well-optimized assays, chemiluminescent reactions can produce a stable output of light for several hours that can be detected using X-ray film or digital imaging equipment, such as a charged-coupled device (CCD). Compared to the other enzymatic methods, chemiluminescence detection is the most widely used and offers the greatest sensitivity, providing pictogram to femtogram-level detection.

When a higher degree of signal amplification and detection sensitivity is needed, poly-HRP secondary antibodies can be used instead of traditional HRP conjugates. The HRP heteropolymer core conjugated to secondary antibodies drastically increases the molar ratio on the secondary antibody without affecting its functionality. This allows more HRP molecules to be available at the immune complex to react with the substrates developing more desirable signals.

Fluorescent Labels

P53 Antibody Western Blot, validation (1:1000 dilution) in T8.

While enzyme-labeled secondary antibodies can be used to detect proteins fluorescently, the use of fluorophore-labeled secondary antibodies is preferred since a substrate development step is not required in the assay. More importantly, fluorophore-labeled secondary antibodies are easy-to-use, quantitative, provide wider ranges of detection and linearity, and have the advantage of multiplexing (i.e., detection of multiple target proteins on a single blot). Secondary antibodies can be conjugated to iFluor® dyes, Alexa Fluor^® dyes, and traditional dyes such as fluoresceins, rhodamines, and cyanines, to generate a detectable single. The target protein is visualized by exciting the fluorophores using an imaging system, such as a ChemiDoc or Odyssey^® imaging system, equipped with an appropriate light source and filter. As the fluorophore absorbs light and transcends to a higher energy state, vibrational effects cause it to relax back to its ground state and emit a photon of light at a longer wavelength. Imaging systems typically use a combination of laser and filter sets, in some cases up to 18 channels, to support dozens of fluorophore pairings.

iFluor® conjugated secondary antibodies exhibit superior brightness and photostability, outperforming Alexa Fluor^® and other spectrally similar secondary antibody conjugates. These iFluor® secondary antibodies recognize IgG heavy chains and all classes of immunoglobulin light chains from either mice, rabbits, or humans. For multiplex western blot analysis, obtain superior images with highly cross-adsorbed iFluor® near-infrared fluorescent dye-labeled secondary antibodies. These antibodies have been manufactured with an additional purification step that helps eliminate species cross-reactivity and increases specificity, and their near-infrared fluorescence minimizes cross-talk.

iFluor® Secondary Antibody Product Guide

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