Signal is very stable and reproducible even after scanning multiple times. Blots were washed, dried and scanned seven times in a row using a SpectraMax Paradigm reader (Figure 6).įigure 6. Proteins were transferred to Immobilon FL and probed with rabbit anti-transferrin for 2 hours, followed by probing with Eu-labeled anti-rabbit IgG for 1 hour. Serial two-fold dilution of transferrin in 1 x sample buffer was loaded on a 4-20% gradient gel and run for 30 minutes. Blots can be scanned multiple times without loss of signal picograms of GST illustrates signal stability after 57 days. Eu-label signal stability and resistance to photo bleaching.įigure 5. Blots can be scanned later as the signal is stable for months. Signal stabilityĪn exceptional feature of the Eu-labels is signal stability and resistance to photo bleaching. Integrated intensities from individual bands shown in Figure 2 analyzed with the SoftMax Pro integrated Excel macro showing total dynamic range of 4 logs and a linear dynamic range of 3 logs. Image of GST dilution series as scanned by SpectraMax Paradigm reader.įigure 3. The system demonstrated sub-picogram detection limit of GST with over 4 logs of positive response of the signal vs. The blot was washed, dried and scanned using SpectraMax Paradigm reader (Figure 2). Proteins were transferred to an Immobilon FL membrane and probed with biotin labeled rabbit anti-GST for 2 hours followed by incubation with ScanLater Eu-labeled streptavidin for 1 hour. A three-fold serial dilution of GST in 1x running buffer was loaded on a 4-20% gradient gel and run for 30 minutes. The sensitivity and dynamic range of the system were tested using glutathione S-transferase (GST). Alternatively, data can be directly exported to ImageJ for analysis. Raw counts can also be directly exported by the software to an integrated Excel macro spreadsheet for analysis and quantification of protein. Images can be optimized and stored in SoftMax ® Pro Software. First, digital photon counting provides TRF counts as raw data which are not altered. By using a microplate detection system, there are distinct advantages. 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.
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