Western blot analysis is susceptible to error and variation in more ways than one, whether it be the technique itself, or the detection chemistry, samples, and materials used in the assay. How do you ensure that the data you generate are accurate and reproducible?
Peer reviewers and journal editors want to know details of study design, combined linear detection ranges, data normalization, controls, and reproducibility of results. Can your imaging system alone give you all the answers?
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“Quantification of gel or blot intensities must be performed with data obtained within a linear range of exposure.”
A wide dynamic range of detection on the Odyssey® CLx and Odyssey Fc Imaging Systems captures data over the entire linear range of your assay in a single image. So, whether you are normalizing target proteins to housekeeping proteins or total protein loading, you can conveniently assess the combined linear detection range and confidently quantitate your results.
“Normalization of signal intensity to total protein loading (assessed by staining membranes using Coomassie blue, Ponceau S or other protein stains) is preferred. “House-keeping” proteins should not be used for normalization without evidence that experimental manipulations do not affect their expression.”
Enzymatic detection (chemiluminescence) can be difficult to control and optimize. Adapt your workflow with near-infrared fluorescence detection and get consistent data each time. Explore your options for multiplex detection and normalization with LI-COR near-infrared fluorescence reagents.
“All images submitted to The Journal of Immunology must accurately represent the original data. Original data (digital files, autoradiographs, films, etc.) for all experiments should be fully annotated, secured, and retrievable.”
Not all image editing software programs are designed to analyze Western blot data. Features in unsupported programs can result in non-linear adjustments, compromising the integrity of your results. Empiria Studio™ Software does not modify raw data, but only changes how data pixels are mapped to the screen.
Consistent training is key to reproducible research results. Train your lab members online with standardized Western blotting protocols, video tutorials, and technical resources at the Lambda U™ Education Portal. Master the foundational concepts and get guidance from Western blotting experts.
Users of Odyssey® Imaging Systems can take advantage of personalized training programs on experimental design, imaging best practices, data analysis support, assay optimization techniques, and methods to meet current Western blot publishing guidelines.