![]() In the indirect detection method, an unlabeled primary antibody is first used to bind to the antigen. This detection method is not widely used as most researchers prefer the indirect detection method for a variety of reasons. With the direct detection method, an enzyme- or fluorophore-conjugated primary antibody is used to detect the antigen of interest on the blot. One common variation involves direct versus indirect detection. Procedures vary widely for the detection step of a western blot experiment. Whatever system is used, the intensity of the signal should correlate with the abundance of the antigen on the membrane. Fluorescent blotting is a newer technique and is growing in popularity as it affords the potential to multiplex (detect multiple proteins on a single blot). Alternatively, fluorescently tagged antibodies can be used, which require detection using an instrument capable of capturing the fluorescent signal. However, digital imaging instruments based on charge-coupled device (CCD) cameras are becoming popular alternatives to film for capturing chemiluminescent signal. The light output can be captured using film. The most sensitive detection methods use a chemiluminescent substrate that produces light as a byproduct of the reaction with the enzyme conjugated to the antibody. Chromogenic substrates produce a precipitate on the membrane resulting in colorimetric changes visible to the eye. Often the secondary antibody is complexed with an enzyme, which when combined with an appropriate substrate, will produce a detectable signal. Most commonly, the transferred protein is then probed with a combination of antibodies: one antibody specific to the protein of interest (primary antibody) and another antibody specific to the host species of the primary antibody (secondary antibody). ![]() Next, the membrane is blocked to prevent any nonspecific binding of antibodies to the surface of the membrane. Subsequently, the separated molecules are transferred or blotted onto a second matrix, generally a nitrocellulose or polyvinylidene difluoride (PVDF) membrane. Nitrocellulose can be blocked easily and will provide a good signal-to-noise ratio.The first step in a western blotting procedure is to separate the macromolecules in a sample using gel electrophoresis. It is resilient and stable and better for protein retention. PVDF is a better choice if you plan to strip and re-probe your blot. It is worth some experimentation with membrane types to identify the membrane that will provide you with the optimal results. Nitrocellulose membranes also have a range of pore sizes on offer, which you can select according to the sizes or protein you’re needing to separate.īiorbyt can guide you to the perfect choice to fit your protocol. The most commonly used membranes are PVDF (polyvinylidene fluoride) and nitrocellulose, both have a range of different versions and your choice will affect your results. Variations in concentration will produce variations in result, it is worth the effort before-hand to obtain clear and accurate results. ![]() Examples are HRP-labeled secondaries used for chemiluminescent detection. In some cases, titration of secondary antibodies is also indicated. ![]() Our data sheets provide a dilution factor range as a guide and we recommend you start with a concentration close to the middle of the range, titrating up or down as required. Biorbyt recommends that antibody titration is carried out each time your conditions are changed. You can experiment with a range of antibody concentrations by varying antibody dilution. Concentration of antibody to antigen, pH and temperature are some of the factors that affect the rate of binding. Select Western blot validated primary antibody specific to your protein of interest.Ĭhoose the correct secondary antibody for detection.īiorbyt are happy to advise you on antibody selection and our secondary antibody selection tool is here to help you.Įstablish the optimum antibody concentration. ![]()
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