Identifying Novel Antigens in Renal Allograft Failure

Renal allografts are needed for patients that suffer renal failure. Patients that receive renal allografts may still experience renal allograft failure caused by immunoresponses that are triggered by antigens. This experiment tries to identify the antigens that are causing these responses. First, proteins are extracted from cells to be used in the sample preparation for an IEF (pH 3-10). The IEF will separate the proteins by their isoelectric point, accounting for the first dimension. The IEF settings will start with 300V 1min, 300V 1hr, 3500V 3hrs, then 3500V 6hrs. The second dimension will involve using the IPG strip and an SDS-PAGE gel (12%). A gel electrophoresis will be run to separate the proteins by size. Once the gel is ready, it is stained using either an Oriole fluorescent stain or a Coomassie Brilliant Blue G-250 stain, and images are acquired. The images are analyzed using Melanie 9 2-D gel software to make sure that the separation was performed correctly and that there are enough proteins for the following steps. The steps mentioned above were performed but require some optimization. The goal is to have enough proteins to transfer to a membrane using immunoblot and then incubating the membrane with serum from patients who have had a renal allograft and then comparing this with healthy controls. Spots of interest will contain proteins that need to be identified. Renal allograft failure has a significant economic and social burden on patients. Identifying alloantigens could help prevent renal allograft failure and save lives.