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10004399A.qxp 9/12/2005 2:08 PM Page i Table of Contents Section 1 Introduction................................................................1 Section 2 Starter Kit Components ............................................1 Section 3 Required Equipment and Reagents ........................1 Section 4 Setup and Operation ................................................2 4.1 4.2 4.3 4.4 4.5 4.6 Overview ..............................................................................
10004399A.qxp 9/12/2005 2:08 PM Page 1 Section 1 Introduction This kit was designed to familiarize you with the MicroRotofor™ Cell before running your own sample. The setup and operation guides you through the assembly and a complete fractionation and harvesting of a mixture of three naturally colored proteins. Note: For a detailed description of the MicroRotofor™ components, the setup, and analysis of the results, please refer to the MicroRotofor™ instruction manual.
10004399A.qxp 9/12/2005 2:08 PM Page 2 • Vacuum source and vacuum trap. (Vacuum in 22–28 mm Hg range) • Deionized water • Pipettes (100 µl – 2.75 ml volumes) • Beaker or equivalent to hold the 3 ml sample volume. Section 4 Setup and Operation Note: For a detailed description of the MicroRotofor™ components, the setup, and analysis of the results, please refer to the MicroRotofor™ instruction manual. 4.
10004399A.qxp 9/12/2005 2:08 PM Page 3 Fig. 1. MicroRotofor components and accessories. MicroRotofor chassis and lid (1), harvesting tray (2), focusing chamber (3), anode assembly (4a), cathode assembly (4b), cathode membrane, (5a), anode membrane, (5b), 10 ml syringes (6), 3 ml syringe (7), forceps (8), assembly tool (9), sealing tape (10), cleaning brush (11), vacuum hose (12), vacuum chamber (13). 1. Rinse the equilibrated ion exchange membranes with deionized water. 2.
10004399A.qxp b. 9/12/2005 2:08 PM Page 4 The cathode assembly (black) is attached to the focusing chamber end containing the cathode membrane (black casing). 4. Align one row of focusing chamber ports with the vents on the electrode assemblies. These are the sample loading ports. (Figure 3) Fig. 3. Alignment of the sample loading ports with the vents on the anode (left) and cathode (right) assemblies. 4.
10004399A.qxp 9/12/2005 2:08 PM Page 5 1. Using the assembly tool as a template, cut two pieces of sealing tape (Figure 4). Position the tape across the template covering all three cutting grooves. Cut the tape with a cutting blade at all three grooves. Each strip of tape can be picked up through the cutouts between two grooves. Fig. 4. Using the assembly tool to measure the appropriate length of sealing tape. 2. Seal the lower row of ports (harvesting ports) with a strip of tape.
10004399A.qxp 9/12/2005 2:08 PM Page 6 Fig. 6. Loading sample into the focusing chamber. 4. When the sample is loaded, make sure that all the channels are filled and that no bubbles remain. Air bubbles will disrupt the electric field, which can lead to poor separation. To dislodge air bubbles from the chamber, tap it gently or aspirate the sample from a channel and load it again. 5. Dry the outside surface of the focusing chamber and seal the row of loading ports with a piece of the sealing tape.
10004399A.qxp 9/12/2005 2:08 PM Page 7 Fig. 7. Placing the focusing assembly onto the anode of the chassis. Fig. 8. Fitting the cathode assembly into the cathode of the chassis. 3. Using a 10 ml syringe, add electrolyte solutions to the electrode assemblies (Figure 9). (Note: The electrode membrane storage solution from the starter kit can be used. Alternatively fresh electrolyte solutions can be prepared) I. Add 6 ml 0.1 M H3PO4 through the vent hole of the anode assembly (red). II. Add 6 ml 0.
004399A.qxp 9/12/2005 2:08 PM Page 8 Note: Keep the vacuum valve closed until the run is completed and you are ready for harvesting the fractions. 8. Turn the power switch to the “ON” position to start the oscillating motor. 9. Set the cooling switch to setting II (20°C). (Note: See Table 3.2 in the MicroRotofor instruction manual, for detailed cooling setting information.) 10. Attach the leads on the lid to a PowerPac HV power supply or other commercial power supply capable of power control at 1 W.
10004399A.qxp 9/12/2005 2:08 PM Page 9 4.5 Power Conditions Table 4.1 lists the recommended power conditions. Power conditions are listed for power supplies capable of maintaining 1 Watt constant power and for power supplies which cannot run under constant power, but are programmable for stepwise constant voltage with limiting current. Note: Refer to the MicroRotofor instruction manual, Tables 3.2, 3.3, & 3.4 for detailed power conditions and cooling setting information. Table 4.
10004399A.qxp 9/12/2005 2:08 PM Page 10 6. Remove the focusing assembly from the running station. First, push focusing assembly towards anode to dislodge the connector from the cathode notch in the chassis. Then lift up gently on the cathode end and remove the anode end (red). 7. With the row of sample loading ports facing up (sealing tape on loading ports removed), position the focusing assembly in the harvesting station (Figure 10).
10004399A.qxp 9/12/2005 2:08 PM Page 11 Fig. 12. Harvesting the fractions. 9. Continue to press down on the focusing chamber for several seconds to aspirate the ten fractions into the harvesting tray. 10. Remove the harvesting tray and turn off the vacuum source. 11. Transfer the fractions to micro tubes or other containers with a syringe or pipet (Figure 13). Fig. 13. Transferring the fractions.
10004399A.qxp 9/12/2005 2:08 PM Page 12 Section 5 Disassembly and Cleaning 5.1 Focusing Assembly 1. Using the assembly tool, loosen and remove the electrode assemblies from the focusing chamber (Figure 14). Fig. 14. Using the assembly tool to remove the anode assembly from the focusing assembly. 2. Using the forceps, remove the ion exchange membranes from the focusing chamber and immediately store them in deionized water or in their respective electrolyte solution.
10004399A.qxp 9/12/2005 2:08 PM Page 13 2. Remove the two screws that secure the Harvesting station to the vacuum block and remove the positioning block, needle array, and needle array holder (Figure 15). Fig. 15. Disassembling the harvesting station (top) and harvesting station components (bottom). 3. Clean and dry the positioning block. 4. Using a wash bottle, wash and rinse the individual needles in the needle array. 5. Clean and dry the vacuum chamber. 6. Inspect and reposition the vacuum gaskets.
10004399A.qxp 9/12/2005 2:08 PM Page 14 Note: For additional information on analysis of results, optimizing protein separation, and troubleshooting, please refer to the MicroRotofor instruction manual.
10004399A.qxp 9/12/2005 2:08 PM Page A Bio-Rad Laboratories, Inc. Life Science Group Web site www.bio-rad.
