Consequently, the resolublization should cover the entire surface of the tube to ensure that the antibody is completely resolublized. Although the lyophilized antibody pellet is often at the bottom of the tube, in some cases the pellet may have localized on the tube wall. Several web sites can be accessed to determine general mRNA expression patterns for specific targets which may serve as a general guideline for target protein expression, although mRNA expression does not always correlate directly to protein expression ( Reconstitution: Care should be taken in re-suspending lyophilized antibodies in order to ensure that the antibody is appropriately resolublized. A literature search should be conducted to ensure that the system that you are using is appropriate for detecting the target protein in question. Tissue/Cell Specificity: Target protein expression is dependent on the cell or tissue being examined. In general this titration should range from 0.2 to 5.0 ug/ml. Why do I see a weak signal or no signal at all on my western blot?Īntibody Titration: A titration experiment should be performed with multiple antibody concentrations to determine the appropriate antibody concentrations for optimal signal/noise ratios and for obtaining the best signal. This is usually prevented in reducing conditions, although interactions can result in the appearance of higher bands. Multimers: e.g., Dimerization of a protein. Relative Charge: The composition of amino acids (charged vs non-charged). Several web sites can be accessed to determine isoforms for specific targets. Expression of different isoforms for the same target protein is highly variable depending on the specific tissue and experimental conditions being used. Isoforms: Many proteins express multiple isoforms that are different sizes. Several web sites can be accessed to determine splice variants for specific targets. Expression of splice variants is highly variable depending on the specific tissue and experimental conditions being used. These splice variants can result in the production of different sized protein products. Splice Variants: Alternative splicing may create different sized proteins produced from the same gene. Post-Translational Cleavage: e.g., Many proteins are synthesized as pro-proteins and then cleaved to give the active form, e.g. Post-Translational Modification: e.g., Phosphorylation, glycosylation etc., which increases the size of the protein. However, migration is also affected by other factors, so the actual band size observed may differ from that predicted. In general, the smaller the protein the faster it migrates through the gel. Western blotting is a technique that separates proteins based on size. Why is the actual western blot band size different from the predicted? The optimal antibody concentration must be determined empirically. Typical antibody concentration range = 0.2 - 5.0 ug/mL, or begin with a 1:1000 dilution of antibody reconstituted at 1 mg/mL. ~25 ug/lane = Amount Cell/Tissue Lysate Protein Used Per Lane Efficient protein of interest transfer is dependent on 1) protein size, 2) transfer power settings, and 3) transfer time.More/longer washing steps can help reduce both specific and nonspecific antibody staining.Always run a positive control to ensure antibody activity (purified protein, transfected lysate, etc.).Increasing/decreasing enzyme substrate incubation time can help amplify/reduce signal, respectively.Increasing/decreasing primary antibody concentration can help amplify/reduce signal, respectively.Western Blotting / Immunoblotting (WB / IB) General Tips to consider when performing a western blot: IP Tips & Tricks | Immunoprecipitation (IP) Protocol.ELISA Tips & Tricks| Enzyme-Linked ImmunoSorbent Assay (ELISA) Protocol.ICC Tips & Tricks | Immunocytochemistry (ICC) Protocol.
IHC Tips & Tricks | Immunohistochemistry (IHC) Protocol.WB/IB Tips & Tricks | Western Blotting/Immunoblotting (WB/IB) Protocol.High Throughput Antibody Characterization Services.Loading Control Antibodies for Western Blot.Enhanced Validation Polyclonal Antibodies.
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