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• LOADING CONTROL WESTERN BLOT SERIES

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.

LOADING CONTROL WESTERN BLOT SERIES

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). Human, Mouse, Rat, Hamster, Xenopus, C.To start a western blotting procedure, gel electrophoresis is used to separate macromolecules in a sample. Human, Mouse, Rat, Canine, Feline, Rabbit, ChickenĪnti-beta Actin Antibody (A121404)Īnti-alpha Tubulin Antibody (A101670) Human, Horse, Cow, Porcine, Chicken, Rat, Mouse The table below shows the most common loading controls for each sample type. It is essential to select a loading control that has a different molecular weight to your protein of interest to ensure that the bands are distinct and expression levels quantifiable. Housekeeping proteins are often chosen as loading controls, including: GAPDH, beta Tubulin, or beta Actin. Loading controls are usually proteins that are constitutively and stably expressed at high levels in the cell type or sample that you are studying. Expression levels of the loading control should not vary between the different sample types. This is essential in order to determine whether the difference in levels of the protein of interest is due to loading variance or sample-to-sample expression level differences. Loading control antibodies are used to confirm that protein loading is the same across a gel. Loading Control Antibodies By Stewart Newlove, PhD