Beckman Coulter PA 800 plus Application Manual
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Beckman Coulter PA 800 plus Application Manual

Beckman Coulter PA 800 plus Application Manual

Pharmaceutical analysis system, sds-mw analysis
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A51970AD
January 2014
Beckman Coulter, Inc.
250 S. Kraemer Blvd.
Brea, CA 92821 U.S.A.
Application Guide
PA 800 plus
Pharmaceutical Analysis
System
SDS-MW Analysis

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Summary of Contents for Beckman Coulter PA 800 plus

  • Page 1 Application Guide PA 800 plus Pharmaceutical Analysis System SDS-MW Analysis A51970AD January 2014 Beckman Coulter, Inc. 250 S. Kraemer Blvd. Brea, CA 92821 U.S.A.
  • Page 2 Made in U.S.A. Beckman Coulter, Inc. grants a limited non-exclusive license to the owner or operator of a PA 800 plus instrument to make a copy, solely for laboratory use, of any portion or all of the online help and electronic documents shipped with the PA 800 plus instrument.

  • Page 3: Revision History

    First Revision, A51970AB, December 2009 Revised corporate address. Second Revision, A51970AC, February 2011 32 Karat Software version 9.1 patch PA 800 plus Software version 1.1 patch PA 800 plus Firmware version 9.2 Numerous syntax and grammatical edits Third Revision, A51970AD, January 2014 Dimension &...
  • Page 4 Revision History 1-iv A51970AD...

  • Page 5: Safety Notices

    Safety Notices Symbols and Labels Introduction The following is a description of symbols and labels used on the Beckman Coulter PA 800 plus Pharmaceutical Analysis System or shown in this manual. WARNING If the equipment is used in a manner not specified by Beckman Coulter, Inc., the protection provided by the instrument may be impaired.
  • Page 6 Safety Notices Symbols and Labels Caution, Moving Parts Label This caution symbol warns the user of moving parts that can pinch or crush. CAUTION PARTS MOVE AUTOMATICALLY A015081L.EPS High Voltage Electric Shock Risk Symbol This symbol indicates that there is high voltage and there is a risk of electric shock when the user works in this area.
  • Page 7 For Beckman Coulter products bearing this label, please contact your dealer or local Beckman Coulter office for details on the take back program that facilitates the proper collection, treatment, recovery, recycling, and safe disposal of this device.
  • Page 8 Safety Notices Symbols and Labels Restriction of Hazardous Substances (RoHS) Labels These labels and materials declaration table (the Table of Hazardous Substance's Name and Concentration) are to meet People's Republic of China Electronic Industry Standard SJ/T11364-2006 "Marking for Control of Pollution Caused by Electronic Information Products" requirements. —...
  • Page 9 Safety Notices Symbols and Labels Alerts for Warning, Caution, Important, and Note WARNING WARNING indicates a potentially hazardous situation which, if not avoided, could result in death or serious injury. The warning can be used to indicate the possibility of erroneous data that could result in an incorrect diagnosis (does not apply to all products).
  • Page 10 Safety Notices Symbols and Labels A51970AD...

  • Page 11: Table Of Contents

    Storing the Assay Components, 1-3 Reagents, 1-3 Materials Required but Not Provided by Beckman Coulter, 1-3 Beckman Coulter Consumables, 1-3 Preparing the PA 800 plus Instrument, 1-4 Installing the Capillary, 1-4 Cleaning the Interface, 1-4 Inserting the Cartridge/Calibrating the PDA, 1-5...
  • Page 12 Contents Running the Assay, 1-12 Launching the SDS MW Instrument, 1-12 Conditioning a New Capillary, 1-13 Running SDS-MW Size Standard and Test Samples, 1-14 Checking the Separation Results, 1-16 Estimating Protein Molecular Weights, 1-16 Troubleshooting Guide, 1-17 CHAPTER 2: Instrument and Detector Initial Conditions, 2-1 Instrument Initial Conditions (All Methods), 2-1...

  • Page 13: Introduction

    This application guide has been validated for use on the PA 800 Enhanced Systems and the PA 800 plus Pharmaceutical Analysis Systems. NOTE The PA 800 plus series systems must be equipped with a photodiode array (PDA) detector to perform this assay. Protein Size Standard The SDS-MW Size Standard contains 10, 20, 35, 50, 100, 150, and 225 kDa proteins.

  • Page 14: Intended Use

    SDS-MW Analysis Kit Intended Use Intended Use The SDS-MW Analysis Kit is for laboratory use only. CAUTION Refer to the Material Safety Data Sheets (MSDS) information, available at www.BeckmanCoulter.com, regarding the proper handling of materials and reagents. Always follow standard laboratory safety guidelines. Materials and Reagents Table 1.1 Contents of this Kit (reorder # 390953) Component...

  • Page 15: Storing The Assay Components

    Upon receipt, store protein Sizing Standard and Internal Standard at 2°C to 8°C. The Capillary, Sample Buffer, and Gel Buffer can be stored at room temperature. If precipitation is noted in the Gel Buffer and Sample Buffer, stir until precipitation is completely dissolved. Materials Required but Not Provided by Beckman Coulter Component Part Number...

  • Page 16: Preparing The Pa 800 Plus Instrument

    How to load and unload trays Installing the Capillary Install a 50 µm I.D. bare fused-silica capillary into a PA 800 plus cartridge set for a total capillary length of 30.2 cm. The SDS-MW assay is optimized using a 30.2 cm capillary with a 20.0 cm effective length from the sample introduction inlet to the center of the detector window.

  • Page 17: Inserting The Cartridge/Calibrating The Pda

    SDS-MW Analysis Kit Sample Preparation Inserting the Cartridge/Calibrating the PDA Insert the cartridge into the system. Close the front panel and calibrate the PDA detector. This procedure should be employed daily or any time the cartridge is replaced. Sample Preparation Preparation of SDS-MW Size Standard Remove SDS-MW Size Standard from refrigerator.

  • Page 18: Preparation Of Protein Sample

    SDS-MW Analysis Kit Sample Preparation Preparation of Protein Sample Desalting the Protein Sample The signal intensity and resolution of this kit are sensitive to the salt concentration in the protein sample. Generally, if the final salt concentration is above 50 mM, the sample loading efficiency will be reduced.

  • Page 19: Non-Reduced Protein Sample Preparation

    SDS-MW Analysis Kit Sample Preparation Add 5 µL of 2-mercaptoethanol. Cap the vial tightly, seal with Parafilm, and mix thoroughly. Heat mixture in a water bath at 100°C for 3 minutes. Place the vial in a room-temperature water bath to cool for 5 minutes before injection. Non-reduced Protein Sample Preparation Comparison of the reduced versus non-reduced state of a protein can yield important structural information.

  • Page 20: Sample Vial Setup

    SDS-MW Analysis Kit Sample Vial Setup Place the vial in a room-temperature water bath to cool for 5 minutes before injection. Sample Vial Setup Before placing the 200 microliter sample vials (or micro vials) into the universal vials, ensure that no bubbles are at the bottom of the micro vials.

  • Page 21: Buffer Vial Preparation And Loading

    SDS-MW Analysis Kit Buffer Vial Preparation and Loading Buffer Vial Preparation and Loading Fill the appropriate number of reagent vials with the SDS-MW Gel Buffer, 0.1 N NaOH solution, 0.1 N HCl solution, and DDI water according to the Tray Configuration paragraphs.
  • Page 22 SDS-MW Analysis Kit Buffer Vial Preparation and Loading Figure 1.2 Fill the Reagent Vials 1. Universal Vial Cap 2. Maximum Fill Level 3. Universal Vials NOTE Carefully fill the buffer vials with SDS-MW Gel Buffer without producing bubbles, within the volume recommended.

  • Page 23: Tray Configuration

    Load the reagents into the system inlet (left) and outlet (right) 6x6 buffer trays using the configuration illustrated in Table 1.3, Inlet Buffer Tray Configuration Table 1.4, Outlet Buffer Tray Configuration. Load the trays into the PA 800 plus system. Inlet Buffer Tray Table 1.3 Inlet Buffer Tray Configuration (Cycle 17-24) (Cycle17-24) (Cycle 9-16)

  • Page 24: Outlet Buffer Tray

    SDS-MW Analysis Kit Running the Assay Outlet Buffer Tray Table 1.4 Outlet Buffer Tray Configuration (Cycle 17-24) (Cycle 17-24) (Cycle 9-16) (Cycle 9-16) (Cycle 1-8) (Cycle 1-8) Waste Gel-S Waste Waste Waste (Cycle 17-24) (Cycle 17-24) (Cycle 17-24) (Cycle 17-24) (Cycle 17-24) (Cycle 17-24) Waste...

  • Page 25: Conditioning A New Capillary

    Figure 1.3 Single Run Acquisition Dialog Box From the field, click on the icon. Method Folder Select the file and specify the data filename of your choice. SDS MW Conditioning - PA 800 plus.met Click on the button and select the option. Arrow Increment A51970AD 1-13...

  • Page 26: Running Sds-Mw Size Standard And Test Samples

    Once the buffer trays and samples have been loaded into the system, you are ready to begin the analysis of the test compounds. NOTE Check the Beckman Coulter web site for the most current versions of pre-programmed sequences and methods for this assay. On the Task bar, select , then .
  • Page 27 SDS-MW Analysis Kit Running SDS-MW Size Standard and Test Samples Figure 1.4 Run Sequence Dialog Box An example of the sequence template pre-programming to run one conditioning run, 24 samples and molecular weight size standards, as well as a shutdown method is shown in Figure 1.5.

  • Page 28: Checking The Separation Results

    SDS-MW Analysis Kit Checking the Separation Results Checking the Separation Results The Protein Size Standard contains seven proteins (10, 20, 35, 50, 100, 150, and 225 kDa). All proteins should be completely separated within 30 minutes using our recommended method. See Figure 1.6 for a typical separation of the Size Standard.

  • Page 29: Troubleshooting Guide

    SDS-MW Analysis Kit Troubleshooting Guide Figure 1.7 Qualitative Analysis Tab for Updating the Size Calibration Curve Troubleshooting Guide Problem Possible Cause Corrective Action Low or unsteady current Capillary Plugged (1) Rinse the capillary with DDI water at 100 psi for 10 minutes and then perform the capillary conditioning method.

  • Page 30: Sample,

    SDS-MW Analysis Kit Troubleshooting Guide Problem Possible Cause Corrective Action Broad peaks or low efficiency Poor capillary end cut Check the capillary end under 10x magnification. Re-cut the capillary end if the cut is not clean or is angled. Improper reduction of sample Reduce sample using recommended procedure. Use fresh mercaptoethanol for sample reduction.

  • Page 31: Chapter 2: Instrument And Detector Initial Conditions

    CHAPTER 2 Method Information Instrument and Detector Initial Conditions The instrument and detector initial conditions are the same for all SDS-MW methods. Instrument Initial Conditions (All Methods) Figure 2.1 Instrument Setup - Instrument Initial Conditions A51970AD...

  • Page 32: Detector Initial Conditions (All Methods)

    This feature facilitates the identification of proteins and other UV absorbing molecules. Capillary Conditioning Method (SDS MW Conditioning - PA 800 plus.met) A new capillary or a used capillary that has been stored for a long period of time must be conditioned using the Capillary Conditioning method before starting CE separation.

  • Page 33: Sds-Mw Separation Method

    Method Information Instrument and Detector Initial Conditions Figure 2.3 Time Program - Capillary Conditioning SDS-MW Separation Method Figure 2.4 SDS MW Separation - PA 800 plus.met Shutdown Method Figure 2.5 SDS MW Shutdown - PA 800 plus A51970AD...
  • Page 34 © 2014 Beckman Coulter, Inc. All Rights Reserved...

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