Western blot technique | rocky ground (2023)

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.

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)

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.

Immunpräzipitation-WB

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.

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)

Secondary antibodies against WB

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.

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

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.

Kits para WB