Master's Thesis Defense - Development of a LED-based Single Color Transilluminator for the Visualization of Fluorescent Stained Proteins in Polyacrylamide Gels
Date: October 1, 2007
Time: 9:00 AM
Location: Bossone Research Enterprise Center, Room: 702
Larry Ross Wilson
Advisor: Peter I. Lelkes, Ph.D.
The analysis of protein interactions that occur is very important even when minimal information exists in the literature, in which case a method of global analysis would be necessary to determine what proteins are major players in the process. Development of a cost effective, versatile system that detects phosphoproteins using fluorescent stains would allow more labs to engage in pathway discovery. Two LED-based illuminators were designed for uniform illumination and cost effectiveness. The two system designed used an offset/hexagonal spacing array and was composed of 83 identical LEDs. The LEDs were arrayed in parallel circuits to reduce voltage requirements. Both illuminators were designed for a 5 V power supply, where LED voltage was reduced by a compensating resistor in series with a set of parallel wired LEDs. The current needed to run the device was 1.5 and 1.7 A for each illuminator, respectively. One illuminator was constructed using high-brightness 525 nm LEDs requiring 3.6 V and another with low-brightness 555 nm LEDs requiring 2.6 V. Uniformity of illumination was a necessary design component and was achieved using an offset/hexagonal LED array configuration and using diffuser screens sufficient to reduce the peaks detected visually and without assistance. Uniform illumination was assessed and determined the most uniform source was the 525 nm illuminator which had an average pixel brightness of 77 (lowest brightness “black” being 0 and highest brightness “white” being 255) and a standard deviation of 9, compared to an average brightness of 70 and standard deviation of 25 for the 555 nm illuminator.
The optimal filter combination was tested with several different filters consisting mostly of Wratten filters and optical long pass filters. The most promising filter was the 603 nm long pass optical filter which allowed visualization of fluorescence, whereas Wratten filters had high optical densities that suited for this application. This device was tested using a Jurkat T-cell model stimulated with pervanadate, a potent protein tyroine phosphatase inhibitor, which leads to a dramatic increase in phosphorylation of proteins, and a dilution series of known protein concentrations to determine detection limit. The 525 nm illuminator was capable of detecting 250 ng of protein and visualizing differences between stimulated and unstimulated Jurkat lysates. The 555 nm device was unable to provide enough output for detection of fluorescent labeled proteins even though the peak emission is equal to the peak excitation for Pro-Q Diamond phosphoprotein gel stain. Comparison was made to a midrange UV illuminator which was capable of detecting 62 ng of protein and visualizing the difference between stimulated and unstimulated Jurkat lysates.
Overall, the combination of a 603 nm long pass filter and a 525 nm illuminator allowed detection and visualization of phosphoproteins stained with Pro-Q Diamond and provides evidence that an LED-based low cost system could be used in labs for quick and accurate phosphoprotein fluorescence detection.
The Bossone Research Enterprise Center is located at the corner of 32nd and Market Streets.