Match the lysis buffer to the target whole cell lysates can often be prepared using a buffer containing NP-40, while extracting cytoplasmic or nuclear proteins may require using Triton X-100 or RIPA buffer, respectivelyĬheck the transfer was performed in the right direction Keep samples on ice prior to adding loading buffer Include protease inhibitors in the lysis buffer, and phosphatase inhibitors if performing phosphoprotein detection Samples contain only low concentrations of the target proteinĬonfirm that an appropriate sample type was used by referring to sites such as UniProt, PAXdb, or, and to antibody manufacturers’ datasheets, for information about protein expressionĭetermine the appropriate amount of sample to load by conducting a linear range experimentĮnrich the target protein through immunoprecipitation, protein precipitation, or sub-cellular fractionation The following table comprises some common western blot complaints and potential fixes, with any application-specific issues being noted in parentheses. While troubleshooting strategies will vary depending on whether a western blot employs chemiluminescent or fluorescent detection, there is considerable overlap between the two methods. Also, optimizing the amount of protein loaded-whether that be the target of interest or a protein used for normalization-is crucial to ensure the amount of protein detected is proportional to the signal on the blot.” Other methods for improving reproducibility focus on using an appropriate imaging system and data analysis software and are included in the table below. A tried and trusted approach is to perform direct detection using fluorescence instead of chemiluminescence, which eliminates the need for a highly variable enzymatic reaction. “Researchers undertaking these types of experiments typically want to quantify their data, making it essential to apply strategies for increasing reproducibility. “A western blot image is far more than just a pretty picture unless a simple yes/no answer is needed,” reports Kristi Ambroz, Senior Director of Science and Global Support, Biotechnology, at LI-COR Biosciences. Search Now Search our directory to find the right western blot products for your research needs. “Additionally, a poor western blot image can mean the expense of having to repeat an experiment or can cause researchers to give up on a set of conditions or reagents that seemingly failed but could have been successful with optimization.” “Such issues can lead to the protein band of interest being incorrectly identified or quantified, translating to inaccurate interpretation of results,” he says. Smeared lanes and saturated bands are other frequent complaints. These include low target protein band intensity, high background, and the presence of multiple bands and/or indiscreet band patterns. Consequences of a poor western blot imageĪccording to Kenneth Oh, Ph.D., Global Senior Product Manager, Applications and Collaborations Protein Quantitation and Imaging Business Unit at Bio-Rad Laboratories, western blot problems fall into several categories. This article lists some common western blot problems and suggests ways of addressing them to achieve more reproducible results. Yet, it can also be one of the more difficult methods to troubleshoot, and a protocol adaptation that improves chemiluminescent western blot data may not be similarly applicable to fluorescent detection. Western blot remains one of the most popular immunoassay techniques for its capacity to provide researchers with a quick, visual representation of their sample.
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