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No matter what the application, Rockland antibodies shine through. The western or immuno blot is a work horse assay for most labs, used to demonstrate the presence or absence of important proteins, detect post-translational modifications, diagnosis is disease and more. Robust bands that identify targets can be visualized after probing your blot with Rockland primary antibodies for detection and conjugated secondary reporting antibodies. Protect your experiments with Rockland antibodies. Compromise elsewhere.
Primary Antibody Reagents
IRDye Antibody Conjugates for Fluorescent Western Blotting
Western Blot Preassembled Kits
The western blot (sometimes called the protein immunoblot) is a widely used method for the detection of specific proteins among a sample of tissue homogenate or extract. Western blot uses gel electrophoresis (SDS-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 followed by another probing with a secondary antibody conjugated to a reporter molecule to visualize the target protein. Primary antibodies conjugated to a reporter do not require secondary antibody visualization. Wash steps with a detergent containing buffer are typically performed after antibody incubations to remove any non-specific binding of the antibodies. Reagents for western blot are available from Rockland as standalone products, but also as easy to use preassembled kits.
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Western blot experiments 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 detecting on a scanner, using film, or a commercial imaging system.
Fluorochrome reporter molecules do not require substrate, but 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 (commonly 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.
Rockland produces hundreds of primary antibodies against a diverse set of targets. Primary antibodies target key protein components of major biological pathways, such as AKT, Notch, Wnt, NFkB and other key signaling pathways. Antibodies are also valuable for drug discovery in the areas of inflammation, infectious disease, neuroscience, and cell proliferation.
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. Alkaline phosphatase is also used, most typically as the reporter in chromogenic western blot assay format.
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.
Streptavidin is a protein purified from the Streptomycesavidinii. The streptavidin tetramers have very high affinity for biotin, and biotin-streptavidin is one of the strongest non-covalent interactions observed in nature. Streptavidin conjugated to enzymatic or flourogenic reporters are used extensively in molecular biology and bio nanotechnology due to the streptavidin-biotin complex's resistance to organic solvents, denaturants, and detergents. Signal amplification is also a benefit of using this reporter system which can improve upon sensitivity when compare to typical antibody-target interactions.
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Rockland produces several luminol based Chemiluminescent substrate for the detection of horseradish peroxidase (HRP). PicoMax™ and FemtoMax™ are designed for high performance in western blotting and is 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 is able 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.
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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 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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Kits may be available for your assay, simplifying your reagent needs. Kits are available for chemiluminescent, fluorescent, and chomogenic assay formats. 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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Rockland Immunochemicals Inc.Gilbertsville, PA 19525E-mail: firstname.lastname@example.orgPhone: 800.656.7625