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The western blot (also called protein immunoblot) is a widely used method for detection of specific proteins among a sample of tissue homogenate or extract. Western blot protocol uses gel electrophoresis (SDS-PAGE or native PAGE) to separate proteins according to the protein molecular weight. The proteins are then transferred from the gel onto a membrane (typically nitrocellulose or PVDF). The membrane is blocked with a protein blocking buffer to prevent nonspecific binding to the antibodies. The membrane is probed using a primary antibody to detect the protein of interest followed by a further incubation with a secondary antibody conjugated to a reporter molecule. The reporter molecule will allowthe visualization of the target protein at the end of the assay. Primary antibodies conjugated to a reporter do not require secondary antibody usage. Wash steps with a mild detergent containing buffer are typically performed after antibody incubations to remove any non-specific binding of the antibodies. Reagents for western blots are available from Rockland as standalone products, but also as easy to use preassembled kits.
IP Western blots provide highly specific results, yet often suffer from heavy/light chain blotting, contamination, and ongoing interference. TrueBlot® products solve nearly all of these problems through increased sensitivity, less background noise, and enhanced accuracy.
TrueBlot® reagents enable you to generate clear, best-quality data in your Immunoprecipitation and Western Blot protocols. Available in several options, from IP Beads alone, to complete IP/Western Blot kits from goat, mouse, rabbit or sheep.
Western blot experiments (much like ELISA immunoassays) can be performed in several formats, most requiring a conjugated secondary antibody to act as the reporter molecule. Reporter molecules include enzymes (peroxidase, alkaline phosphatase) and fluorochromes.
Enzymes require substrate that allows for color or light to be generated. The data can be detected on a scanner, x-ray film, or a commercial imaging system, often referred to as a multiplexer. Multiplexing is the ability for multiple types of information to be transferred utilizing a single information medium.
Fluorochrome reporter molecules do not require substrate, but, because of their chemiluminescence, they do require specialized equipment with light filters for data collection. Western blot experiments immobilize protein analyte onto a membrane. The choice of membrane depends on the type of experiment to be performed. Most commonly used are nitrocellulose or polyvinyldifluoride (PVDF). Nitrocellulose is easy to use and provides suitable data for most common enzymatic reporter experiments. Low fluorescent PVDF membranes are recommended for fluorescent western blot applications. A schematic illustration of 2 common western blot formats is shown in the figure to the left.
Secondary antibody conjugates are ideal for western blotting. When choosing a secondary antibody conjugate for an antibody assay consideration must be given to target species, conjugate (i.e. peroxidase, FITC, Biotin) and host species. In addition to standard secondary antibodies, Rockland offers pre-adsorbed secondary antibodies which are suitable for detection methods where cross-reactivity may be an issue.
Reporter enzymes are used extensively in molecular biology because they allow visualization or detection of immune complexes. Horseradish Peroxidase (HRP) is a widely used reporter enzyme, and depending on the substrate it can yield a chromogenic, or luminescent product (chemiluminescence). Alkaline phosphatase is also used, most typically as the reporter in chromogenic western blot assay format.
Antibodies Conjugated to Alkaline Phosphatase (AP or Alk Phos) are used in the detection of proteins in Western blotting and ELISA immunoassay procedures. The alkaline phosphatase (AP) catalyzes colorimetric reactions using BCIP/NBT Substrates or FemtoMax chemiluminescent substrate. Secondary Antibody conjugates are conjugated to the highest grade of alkaline phosphatase using Rockland’s proprietary technology.
Rockland conjugates a broad group of secondary antibodies to many of the classic and next generation of fluorescent markers including fluorescein, Texas Red, Phycoerythrin. Rockland also produces many next generation flurochrome dyes. These are designed for detection of primary antibodies in multiplex, multi-color analysis. Next generation fluorochrome conjugates (Atto-tec dyes, DyLight dyes) offer superior absorption (high extinction coefficient), high fluorescence quantum yield and superior high photostability. All of the conjugates are ideal for various immunofluorescence based assays including fluorescent western blotting, immunofluorescence microscopy, FLISA, and more.
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Fluorescence technology is widely used to detect proteins. However, many common visible fluorophores often result in considerable background fluorescence in the visible range. Visible fluorophores are rarely used for membrane-based protein detection because of this high background. IRDye® 800 antibody and reagent conjugates are specifically designed for protein detection methods that use longer-wavelength, near-infrared (IR) fluorophores to visualize proteins in western blotting and other immunoassay applications. Very low background fluorescence in the IR range provides for a much higher signal-to-noise ratio than visible fluorophores. Detection levels in the picogram range on Western blots rival the sensitivity of chemiluminescence on film. IRDye® 800 conjugates are optimized for the Odyssey® Infrared Imaging System developed by LI-COR. IRDye® 800 conjugates are also suitable for immunofluorescence microscopy using commercially available excitation/emission filters in the 780nm/820nm range. Dual simultaneous labeling in western blots or microscopy is achieved when IRDye® 800 conjugates are used in conjunction with IRDye® 700 conjugates. IRDye® 800 conjugates provide an ultra-sensitive and convenient alternative to standard chemiluminescent protein detection methods, as well as a valuable tool for multicolor imaging.
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Fluorescent Western Blot Buffers
Rockland produces several luminol based substrates with chemiluminescence for the detection of horseradish peroxidase (HRP). PicoMax™ and FemtoMax™ are designed for high performance in western blotting and are functional on both nitrocellulose and PVDF membranes. FemtoMax™ produces chemiluminescence and allows for the detection of down to femtogram (10-15) amounts of antigen. Detection methods may include photographic film or other imaging methods, including highly sensitive CCD camera based systems.
Chromogenic blotting substrates are available from Rockland in a variety of specifications and formats. The appropriate substrate choice depends on the enzyme label, desired sensitivity and form of signal or method of detection needed.
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When performing a western blot, the blocking buffer should not be overlooked. The blocking buffer fills in the locations on the membrane that can still bind protein and cause background if not treated. The critical reagent in a blocking buffer is protein, where the protein is non-antibody reactive. Popular blocking proteins include non-fat dried milk (NFDM), BSA, casein, and combinations thereof. Of note, a single blocking agent may not be sufficient for all western applications. Some blocking agents can interfere with primary antibody activity, or may not be compatible with the reporter system in use, or produce undesired auto-fluorescence. Rockland develops several blocking buffer reagents suitable for all western blot applications, including BLOTTO-NFDM and BSA for standard applications, and a specially formulated Blocking Buffer for Fluorescent Western Blotting.
Read more on Rockland's Blocking Buffer Formulations
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Western blot kits may be available for your assay, simplifying your reagent needs. Rockland offers Kits for chemiluminescence, fluorescent, and chromogenic immunoassay formats. Our western blotting kits are configured with simple and easy to use protocols for both beginner and expert users alike. Kits are species specific for detection of mouse or rabbit primary antibodies, and come ready as a format specific package that includes membrane blocking reagent, washing buffers, secondary antibodies and substrate (if required). Some of our more popular kits include FemtoMax kits for chemiluminescent applications, and IR™dye or DyLight™ kits for detection on fluorescent western blot protocol imaging systems.
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Western Blot or immunoblot is a work horse immunoassay for most labs, used to demonstrate the presence or absence of important proteins, detect post-translational modifications, diagnose disease and more. Robust bands that identify targets can be visualized after probing your western blot with Rockland primary antibodies for detection and conjugated secondary antibodies. Rockland has also developed Revitablot™ Western Blot Stripping Buffer, which contains solutions in a proprietary combination to enhance the removal of bound antibodies from western blot membranes for repeated use. The proprietary formulation of the solution ensures high stripping efficiency with low backgrounds.
Revitablot™ Western Blot Stripping Buffer(500mL) is a uniquely formulated, ready-to-use reagent specifically designed for western blotting. Revitablot™ is faster and more efficient at stripping primary and secondary antibodies, allowing blots to be stripped multiple times for the repeated use of membranes. This solution can be used at room temperature and only requires 5-20 minutes of incubation time to strip the membrane. Revitablot™ Western Blot Stripping Buffer(50mL) is gentle and effective on both nitrocellulose and PVDF western blot membranes.
Loading controls are important for correct interpretations of western blots. Control Antibodies are used to normalize the levels of protein detected by confirming that protein loading is the same across the gel. The expression levels of the loading control should not vary between the different sample lanes.
Popular Loading Control Antibodies for Western Blotting:
Many of Rockland Immunochemicals’ antibodies have been validated for use in In-Cell Western (ICW) assays. Since ICW assays are related to immunofluorescence most antibodies that have been validated and approved for these aforementioned immunoassays can be similarly used with ICW assays. Antibodies suitable for cell based immunoassays can be identified under the applications field of the Rockland website catalog.
Read more about In-Cell Western Assays Here.
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