This application requests funds to purchase a Protein Simple Jess automated capillary electrophoresis and western blotting system. This instrument performs separation of proteins by capillary electrophoresis followed by in-gel immunoblotting with chemiluminescent or fluorescent detection of immunolabeled bands. Normalization using in-gel fluorescent labeling of all proteins is also available. The user loads samples and primary and secondary antibodies into a multiwell plate. All other steps from protein loading to protein separation, immunodetection and image acquisition are done automatically. Specific advantages of this technology include the ability to obtain western blotting and normalization data for 24 samples in under 4 hours, elimination of the variability inherent in electro-transfer of protein from the electrophoresis gel to immunoblotting membranes, and very high sensitivity (less than 1.5 micrograms protein is required per lane). As a result, inter-assay variability approaches 15% and throughput is increased. This technology is thus well suited to experiments requiring that large numbers of samples and/or antibodies be run. A growing number of antibodies have been validated in this system, with over 1,400 currently listed on the manufacturer?s website. The instrument will facilitate research in several areas. In spinal cord injury research, it will be used for ongoing analysis of changes in intracellular signaling elicited by functional electrical stimulation in ongoing research in both mice and human subjects, and to elucidate mechanisms for changes in functional outcomes observed with genetic manipulation or candidate drugs. In traumatic brain injury research, this technology will be invaluable in investigating a growing number of proteins linked to vascular and behavioral abnormalities following repeated exposures to mild blast waves. In ongoing Alzheimer?s disease research, protein levels of key factors identified through an ever-growing range of genomics techniques aimed at understanding the biological basis for the strong link between apolipoprotein epsilon 4 and cognitive impairment will be interrogated. New research investigating new drug candidates to mitigate effects of depression is also expected to rely heavily on the capabilities of this systems. Among minor users, the instrument will be used to accelerate progress on research on multiple aspects of cancer biology in cell culture systems that relies continuously on western blotting for a wide variety of endpoints and, in cardiovascular research, to enhance research that focuses on intracellular stress signaling pathways converging on hypoxia-inducible factors which utilizes western blotting to characterize activation states of such pathways by western blotting.
This application requests support for an instrument that uses a relatively new technology for quantifying proteins in cells, tissues or solutions that offers several advantages over currently employed methods used in laboratories at James J Peters VA (JJPVA) including greater speed, reproducibility and accuracy. Availability of the instrument to JJPVA investigators will accelerate the pace of progress, rigor and productivity of research on common health problems that are high priorities for VA research including traumatic brain injury, Alzheimer?s disease and other mental health issues, cancers common among veterans such as lung and prostate cancer, aging biology and spinal cord injury. The accelerated pace of research afforded by this new technology will hasten the discovery of new knowledge that could ultimately improve treatments, health or quality of life of Veterans afflicted by these medical issues.