WESTERN BLOT QUANTIFICATION IMAGEJ BACKGROUND PRO

SoftMax Pro Software analysis of western blot dataĪll experiments were conducted using the ScanLater Western Blot Detection System for either the SpectraMax i3 or SpectraMax Paradigm Multi-Mode Microplate Reader. The new ScanLater™ Western Blot Detection System is a simple, sensitive, and stable platform that provides excellent protein analysis capability in a multi-mode plate reader. This stability enables repeat reading of membranes allowing more accurate quantitation.

Therefore, the signal remains stable for weeks to months. The method does not involve enzyme detection, and the Eu-chelates are resistant to photo bleaching. There is no camera blooming which is often seen with chemiluminescence or standard fluorescence detection thus the system provides sharp bands and superior image quality. This significantly reduces background from auto-fluorescence or other sources of short lifetime emissions. Images are generated utilizing time resolved fluorescence (TRF) mode detection of Europium (Eu) which has a 1 ms fluorescence lifetime. Detection with ScanLater TRF Western Blot Detection Cartridge (4). Eu-labeled ScanLater Secondary Antibody (3) binds to primary antibody. Use existing primary antibody (1) for binding to protein of interest (2). Membranes are incubated with Europium-chelate labeled secondary antibodies or streptavidin that bind specifically to the primary antibody bound to the protein of interest (Figure 1).įigure 1. ScanLater ® Western Blot System workflow follows standard gel loading and blotting methods up to the secondary antibody incubation step. In addition, a comparison of Scanlater Western Blot System to a chemiluminescence method demonstrates improved sensitivity. We demonstrate extended dynamic range due to reduced background as well as stability of detection over time and number of reads. Here we report on a novel system for western blot membrane protein analysis that is incorporated into SpectraMax ® i3 and SpectraMax ® Paradigm ® Multi-Mode Microplate Readers. However, each technique has its limitations, and there is a continuing need to improve quantitation, accuracy, and dynamic range. Various techniques are used to detect proteins on western blot membranes including fluorescence and chemiluminescence. Protein detection is important for pharmaceutical and clinical research today, and western blots are among the most common methods employed for this purpose.

Western blot quantification imagej background