Isolation, Purification and Protein Content of Pancreatic ...

the Pancreapedia entry by Dingsdale et al (11).1. Materials for Isolation of ER1.1 Rat or mouse pancreas as desired fromcontrol or treated animals.1.2 Motor driven Potter – Elevjhem homogenizer.The glass Teflon homogenizer can be purchasedfrom Kontes, Wheaton or Charles Thomas. Weuse a Charles Thomas Size B or Kontes size 22for rat or mouse pancreas and a Kontes size 20for isolated acini. For homogenizing intactpancreas first mince the tissue with scissors toenhance tissue breakdown. We then used aserrated pestle to prepare the homogenate. Themost important aspect is the clearance whichgenerally increases with the size of the chambervolume. The Kontes homogenizers generallyhave a smaller clearance. A Polytron typehomogenizer should not be used because it willdisrupt cellular organelles and fragment all cellularmembranes.1.3 A high speed refrigerated centrifuge and anultracentrifuge. We use Beckman ultracentrifugeswith a Ti70 angle head and a SW-41 swingingbucket rotors with appropriate tubes.1.4 A 2M sucrose stock for preparingdiscontinuous sucrose gradients.1.5 A homogenizing solution of 0.3 M sucrose, 10mM Hepes-NaOH (pH 7.0 at 4 0 C) with 2mMdithiothreitol and protease inhibitors usually 1mMbenzamidine, 0.5 mM PMSF (freshly added froma 100 mM stock in ethanol) and 150-200 units/mlof aprotinin, a kallikrein inhibitor. For studies ofprotein phosphorylation, kinase and phosphataseinhibitors can be added.1.6 A solution of 1 M KCl and 1 mM puromycin forremoving ribosomes.2. Methods for Isolation of ER2.1 Rat or mouse pancreas is homogenized in 5-10 volumes of ice-cold homogenizing solution byuse of a motor driven glass Teflon homogenizer atmedium speed. For pancreas 7-10 strokes over 1min at about 1000 rpm is usually sufficient. Forisolated acini, the acinar suspension iscentrifuged at low speed (50-100 g) and washedin either calcium free KHB or PBS andresuspended in 10 volumes homogenizingsolution. Homogenization requires 15- 25 strokesof the motorized pestle.2.2 The homogenate is first centrifuged at 500-1000 g for 10 min and the supernatant thencentrifuged at 12,000 g for 10-20 min to removeall debris, nuclei, zymogen granules andmitochondria. If you set the final spin at the highend of the g x time range ( 12,000 g for 15 min)you will remove mitochondria more completely,but will lose some RER.2.3 To collect vesiculated ER also referred to as“microsomes”, one of the following procedures isused.a. Centrifugation at 150,000 g for 30 min or100,000 g for 60 min in the Ti 70 rotor orequivalent to obtain total microsomes. (Note1)b. Centrifugation at 27,000 g for 10-20 min toobtain a heavy microsomal fraction whichcontains primarily RER (17). This method hasthe advantage of speed but is less pure thanmethod c.c. Centrifugation at 100,000 g for 60 min in a SW27 or SW 41 rotor of a gradient prepared bylayering 4 ml of the postmitochondrialsupernatant on top of a 2ml cushion of 1.3 Msucrose buffered with 10 mM Hepes (pH 7.0)and supplemented with 1 mM MgCl 2 , 2 mMDTT and protease inhibitors. Purified roughmicrosomes are collected at the bottom of thetube (22).2.4 Purification of both RER and SER is by theworkflow shown in Fig 1. The total microsomalpellet prepared as in 2.3a is resuspended in 1.25M sucrose, 20 mM Hepes with benzamidine, DTTand PMSF homogenizing solution (see point 1.5)2