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Katie Mills, Marko Radic, Indira Neeli, Margaret Phillips, Carolee Cutler Peck, Byron Wilkes, James Fleming, Alessandro Iannaccone, Natalie Kerr; Protocol for Protein Isolation from Human Orbital Fat Tissue: Use for Western Blots and Antigen Biomarker Identification in Thyroid Eye Disease. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5903.
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© ARVO (1962-2015); The Authors (2016-present)
To develop a protocol for isolating proteins from orbital adipose tissue of patients with Thyroid Eye disease (TED) and unaffected controls to test the hypothesis that TED patients with increased orbital fat volume present with serum auto-Abs directed against orbital fat tissue antigens that can be used as potential disease biomarkers in TED.
Sample Collection: Human orbital fat tissue (hOFT) was harvested from 3 control subjects unaffected by TED (hOFT-CTRL) during planned eyelid and orbital surgeries in which orbital fat is routinely collected and discarded by surgeons at Hamilton Eye Institute. The orbital fat was collected from the operating room and stored in aliquots at -80οC. Phase Separation: The frozen adipose tissue was homogenized with 5mL Trizol Reagent and a Power Homogenizer. The samples were centrifuged for 10mins at 2-8οC at 12,000 xg. The top layer was discarded. The resulting liquid layer was isolated and the remaining pellet discarded. 1mL chloroform was added to the sample and centrifuged at 12,000 xg at 2-8οC for 15 minutes. The result was 3 layers, 2 liquid and one interface. The adipose tissue proteins were contained in the interface and lower layer. Acetone (3x volume of the sample) was added to the isolated interface and lower layer, mixed and centrifuged at 10,000 rpm for 10 minutes. The acetone was discarded without disturbing the resulting pellet. The pellet was washed with additional Acetone and dried. The protein pellet was mixed with 50UL 1x SDS gel loading buffer and boiled to facilitate suspension. The sample was stored at -80οC. Electrophoresis: The sample was mixed with 40uL of lysate and loading dye mix. The samples were loaded and ran for 1 hour at 35 milliAmps with a wide range BRL protein marker.
By following this protocol, hOFT proteins were successfully isolated from hOFT intraoperative samples with little apparent degradation.
With a successful protein isolation protocol, Western Blots to test for presence of auto-antibodies against orbital fat tissue antigens can be performed. The comprehensive analysis of the orbital fat tissue proteome from TED and control individuals and the identification of autoantibodies from patients and controls will provide testable hypotheses to explore TED pathogenesis.
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