Maximize the target's concentration by enriching the sample beforehand. The target protein is not found in high concentrations in your sample. Use a positive control and a molecular weight marker matched to the size range of the target protein. Reduce washing step repetitions or duration. Secondary antibody is inhibited by sodium azide.ĭo not use sodium azide with HRP-Conjugated antibodies. Ensure you are using the correct transfer times. Make sure the transfer apparatus is set up correctly. Make sure detection reagents are functional by testing with a different primary antibody. Increase the blots incubation time with detection reagent. Incubation with detection reagent not sufficient. Use fresh antibody (the effective concentration is lowered after each use). Use protease inhibitors and run the recommended positive control. Load a larger amount of protein onto the gel. Utilize a higher concentration of antibody and or incubate for a longer period (i.e. Insufficient primary or secondary antibody has bound to the protein of interest. if your primary is rat, be sure you are using an anti rat secondary). The primary and secondary antibodies are not compatible.Įnsure you are using secondary antibody that binds to your primary antibody (i.e. And finally, in fluorescence, a fluorophore-labeled antibody emits the signal.īioLegend Western Blot Troubleshooting Guide In a chemluminescence-based method, light is emitted by the reaction shown. The colorimetric method detects signal via colored precipitate. There are multiple ways to detect proteins of interest in a western blot. These fluors typically emit in the near infrared range for detection.įigure 4. For fluorescence-based detection methods, fluorophore-conjugated primary antibodies are used. Direct-Blot™ products allow users to utilize primary antibodies directly conjugated to HRP, avoiding the need for a secondary reagent and streamlining the western blot process. Enzyme reporters like alkaline phosphatase (ALP) and horseradish peroxidase (HRP) are then used to generate signal in combination with a substrate like our Western-Ready™ ECL Substrate Kit. In colorimetric and chemiluminescent detection methods, the membrane is subsequently incubated with a detectable tagged secondary antibody specific to the host species of the primary antibody. After transfer, the membrane is incubated with primary antibodies that bind specifically to the target protein, the primary antibody is not typically directly detectable. This is why conformation epitope-specific antibodies and even flow cytometry antibodies may not always work in a western blotting assay. It is important to recall that SDS treatment of samples denatures proteins, causing them to lose their native conformation. BioLegend offers the Prime-Step™ Prestained Broad Range Protein Ladder, a three-color protein standard with 10 pre-stained chromophore-conjugated recombinant proteins covering a wide range of molecular weights, from 6.5 to 270 kDa. By using a ladder, the size of proteins in the sample lanes can easily be determined. Protein samples are run along side a protein ladder containing several standards of known molecular weights. Once an electrical field is applied to the gel, small protein molecules move quickly the through the gel matrix toward the positive electrode, while larger proteins move through more slowly, resulting in a series of bands containing proteins of a particular size (Figure 2). Because of the SDS, all proteins will have the same negative charge, resulting in separation being based on size rather than charge. SDS is a type of detergent that adds a negative charge to amino acids in a protein, this along with heat applied during sample prep disrupts the tertiary and secondary structure of the protein. The most common type of electrophoresis used is called SDS-PAGE (SDS-Polyacrylamide Gel Electrophoresis). To start a western blotting procedure, gel electrophoresis is used to separate macromolecules in a sample.
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