When it comes to protein analysis, the Western blot is one of the most widely used techniques. It can be used to identify, quantify and characterize proteins in a sample and is essential for identifying post-translational modifications and protein-protein interactions. However, the Western blot can be inaccurate if the conditions are not properly optimized or if the samples are not well prepared. This link bosterbio.com

Often, errors in the western blot are caused by incorrect protein transfer from gel to membrane or by non-specific binding. The best practice is to use a pre-mixed blocking reagent for your specific antibody to avoid any non-specific binding. It is also recommended to run a dilution curve on the membrane and gel to ensure that the protein of interest is within its linear detection range. In addition, the blot should be run in the most appropriate percentage of gel to prevent any over-dissolving of the protein and any interference from the other proteins present.

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Other error sources can be related to the method of sample preparation. For example, excessive thawing and refreezing of tissue or cell lysates can affect the quality of proteins by disrupting their structure. Samples should be centrifuged or fractionated as soon as possible after sampling to reduce degradation by endogenous proteases. In addition, it is important to avoid repeated freeze/thaw cycles as this can lead to aggregation and loss of the target protein.

Another common source of errors in the Western blot is over-incubation of the primary antibodies. It is therefore important to optimize the incubation time of the primary antibody by running a dilution curve. In addition, it is recommended to use high-quality secondary antibodies that are labeled with a fluorescent or chemiluminescent tag to increase the sensitivity of the signal. Finally, it is important to use a good quality digital imager that allows you to accurately quantify the results of your protein experiments. To find the right system for your needs, request pricing today by filling out this form and a product expert will be in touch.