western blot quantification imagej and ccd imager

Want to learn more? Contact LI-COR today and let us help choose the right detection and imaging methods for you.The Trans-Blot Turbo system is a rapid protein transfer apparatus that can transfer protein to membrane in as little as 3 minutes. There are many other important aspects to chemiluminescence, x-ray film, and digital imaging. When switching to NIR fluorescence entirely, imagers like the Odyssey DLx have the complete capacity of NIR imaging and can be versatile tools for imaging not only Western blots but plate-based assays, slides, and excised organs as well.Ĭheck out the Advanced Applications Brochure for additional information. To eliminate these factors completely, consider transitioning to NIR fluorescence with imagers like the Odyssey XF, which can still use current chemiluminescence protocols. However, the limitations of enzymatic detection are inherent to chemiluminescence. The C-DiGit scanner, for example, is a practical and useful option for chemiluminescent research. LI-COR can help mitigate x-ray issues with digital imaging. Transition to Near-Infrared (NIR) Fluorescence It can facilitate data analysis not only for an individual experiment but between researchers as well. Unlike other imagers, the Odyssey XF does not introduce image manipulation, such as binning and stacking, during image acquisition. With the largest dynamic range of any available chemiluminescence digital imager, it captures the strongest and faintest bands in a single image without saturation. With a larger dynamic range than film, the C-DiGit scanner can capture more data in an acquisition and minimizes the need for multiple exposures.įor a digital imager capable of both chemiluminescence and near-infrared fluorescence (NIR), utilize the Odyssey ® XF. For chemiluminescent research, the C-DiGit ® Blot Scanner is useful because it eliminates the need for a darkroom along with film and developing reagents expenses. Options to Considerĭepending on the nature of an experiment, there are various digital imaging options that can help generate consistent and credible results. Additionally, its wider dynamic range can collect more data and therefore decrease the loss of very strong and weak bands. Digital imaging is useful because it captures more data than x-ray film in a single exposure and reduces the need for multiple exposures. LI-COR can help bypass any potential x-ray film constraints by facilitating a transition to digital imaging. It is therefore important to understand the variability of ECL in its entirety before proceeding with experimentation. Overall, these factors make it difficult to achieve precise quantification. Additionally, chemiluminescence is unable to multiplex signals or simultaneously detect two targets with the same molecular weight in a single sample that is on the same blot. This makes it challenging to accurately quantify the protein abundance on the membrane. Ultimately, it can be difficult to capture the enzymatic reaction for each target simultaneously and at the same point each time. Low temperature reduces enzymatic activity, so substrates should be equilibrated at room temperature.Signal intensity peaks and fades as the reaction slows over time.Substrate can be depleted by strong bands with high enzyme concentration.Substrate type affects signal stability due to differing reaction rates.Substrate availability affects signal intensity.While chemiluminescent detection is widely used, there are certain aspects to keep in mind when considering it as a Western blotting method. The light photons are then measured with one of two detection methods: x-ray film or digital imaging.įor more information, check out the “Introduction to Western Blotting” learning path in the Lambda U ® education portal. Consequently, HRP causes the substrate to oxidize and transiently produce a signal in the presence of hydrogen peroxide (H 2O 2). The most common chemiluminescent detection substrate, HRP, is luminol-based. The blot is then incubated with substrate to generate a signal, or a production of light. For its enzyme, ECL typically uses horseradish peroxidase (HRP) or alkaline phosphatase (AP). The secondary antibody, which is labeled with an enzyme, binds to the primary antibody. How it WorksĬhemiluminescent Western detection begins when the primary antibody first recognizes the target protein or antigen on the membrane. A distinguishing characteristic is the use of an enzymatic reaction for protein detection. Enhanced chemiluminescence (ECL) or chemiluminescent Western detection is a traditional method for conducting Western blotting research.

Western blot quantification imagej and ccd imager