Isolation, Purification and Protein Content of Pancreatic ...

Fig 2. Following the purification of rat pancreatic RER by immunoblotting of known protein. Western blots ofselected proteins in pancreatic lysate (total) a 14,000 g pellet (P1) a subsequent 150,000 g pellet (P2), RERpurified on the sucrose gradient described in Fig 1, and RER stripped with puromycin and high salt. Calreticulinand BiP are content markers for the RER, S6 a ribosomal protein, Cyclophillin A a cytosolic marker and VDAC1 amitochondrial marker. Modified from Ref (7).2.7 Alternative methods of ER fractionation thathave been applied to the rodent pancreas includecontinuous manitol density gradients (3) andPercoll gradients (9). For integration into anearlier scheme of cell fractionation used forguinea pig pancreas see Tartakoff and Jamieson(19). Purification of cat pancreatic microsomesand characterization by enzymatic markers isdescribed by Milutinovic et al (15) and Kribben etal (13). Preparation of canine microsomes andtheir use for protein translation studies is detailedin Walter and Blobel (21).3. Analysis of ER ProteinsAnalysis of the protein composition of thepancreatic ER has been primarily carried out byN-terminal Edmean sequencing (5),immunoblotting and immunohistochemistry (6) ofindividual proteins and by high throughputproteomic analysis. Both immunoblotting andmass spectrometry are dependent on the purity ofthe preparation, although immunohistochemistryhas the ability to localize without purification andis therefore an excellent tool for confirmationwhen antibodies exist. It also allowsdetermination of the cell type containing theprotein in the pancreas. One area that has beenstudied extensively using canine pancreatic ER isthe protein translocation and biosynthesismachinery (10, 22) which has led to theidentification of the individual proteins involved.Recently, proteomic techniques have beenapplied to the pancreatic ER. Two large datasetsas well as several more targeted studies have4