Beckman Coulter PA 800 plus User guide

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

Application Guide
PA 800 plus Pharmaceutical Analysis System
SDS-MW Analysis
PN A51970AD (January 2014)
© 2009-2014 Beckman Coulter, Inc. All rights reserved.
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.
Beckman Coulter and the stylized logo are trademarks of
Beckman Coulter, Inc. are registered in the USPTO.
All other trademarks, service marks, products, or services
are trademarks or registered trademarks of their
respective holders.
Find us on the World Wide Web at:
www.beckmancoulter.com
Printed in U.S.A.

A51970AD iii
Revision History
Initial Issue, A51970AA, April 2009
32 Karat Software version 9.1
PA 800 plus Software version 1.1
PA 800 plus Firmware version 9.0
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 & instruction edit

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1-iv
Revision History

A51970AD v
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.
General Biohazard Symbol
This caution symbol indicates a possible biohazard risk from patient specimen contamination.
Caution, Biohazard Label
This caution symbol indicates a caution to operate only with all covers in position to decrease risk
of personal injury or biohazard.

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vi
Safety Notices
Symbols and Labels
Caution, Moving Parts Label
This caution symbol warns the user of moving parts that can pinch or crush.
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.
Class 1 Laser Caution Label
A label reading “Complies with 21 CFR 1040.10 and 1040.11 except for deviations pursuant to Laser
Notice No. 50, dated June 24, 2007” is found near the Name Rating tag. The laser light beam is not
visible.
CAUTION
PARTS MOVE
AUTOMATICALLY
A015081L.EPS
144557-AB
A016352L.EPS

A51970AD vii
Safety Notices
Symbols and Labels
Sharp Object Label
A label reading “CAUTION SHARP OBJECTS” is found on the PA 800 plus.
Recycling Label
This symbol is required in accordance with the Waste Electrical and Electronic Equipment (WEEE)
Directive of the European Union. The presence of this marking on the product indicates:
1. The device was put on the European Market after August 13, 2005.
2. The device is not to be disposed of via the municipal waste collection system of any member
state of the European Union.
It is very important that customers understand and follow all laws regarding the proper
decontamination and safe disposal of electrical equipment. 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.
Disposal of Devices Containing Mercury Components
This product contains a mercury-added part. Recycle or dispose of according to local, state, or
federal laws. It is very important that you understand and comply with the safe and proper disposal
of devices containing mercury components (switch, lamp, battery, relay, or electrode). The
mercury component indicator label can vary depending on the type of device.
CAUTION
SHARP OBJECTS
A16558-AA
A016351L.EPS

A51970AD
viii
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.
China RoHS Caution Label — This label indicates that the electronic information product contains
certain toxic or hazardous substances. The center number is the Environmentally Friendly Use
Period (EFUP) date, and indicates the number of calendar years the product can be in operation.
Upon the expiration of the EFUP, the product must be immediately recycled. The circling arrows
indicate the product is recyclable. The date code on the label or product indicates the date of
manufacture.
China RoHS Environmental Label — This label indicates that the electronic information product does
not contain any toxic or hazardous substances. The center “e” indicates the product is
environmentally safe and does not have an Environmentally Friendly Use Period (EFUP) date.
Therefore, it can safely be used indefinitely. The circling arrows indicate the product is recyclable.
The date code on the label or product indicates the date of manufacture.

A51970AD ix
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).
CAUTION
CAUTION indicates a potentially hazardous situation, which, if not avoided, may
result in minor or moderate injury. It may also be used to alert against unsafe
practices. The caution can be used to indicate the possibility of erroneous data
that could result in an incorrect diagnosis (does not apply to all products).
IMPORTANT IMPORTANT is used for comments that add value to the step or procedure being performed.
Following the advice in the IMPORTANT notice adds benefit to the performance of a piece of equipment
or to a process.
NOTE NOTE is used to call attention to notable information that should be followed during installation, use,
or servicing of this equipment.

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Safety Notices
Symbols and Labels

A51970AC xi
Contents
Revision History, iii
Safety Notices,v
Symbols and Labels,v
CHAPTER 1: Introduction,1-1
Protein Size Standard,1-1
Internal Standard,1-1
Intended Use,1-2
Materials and Reagents,1-2
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
Sample Preparation,1-5
Preparation of SDS-MW Size Standard,1-5
Preparation of Protein Sample,1-6
Desalting the Protein Sample,1-6
Protein Sample Concentration,1-6
Reducing the Protein Sample,1-6
Non-reduced Protein Sample Preparation,1-7
Preparing Alkylation Reagent,1-7
Preparing the Sample,1-7
Sample Vial Setup,1-8
Buffer Vial Preparation and Loading,1-9
Preparing the Reagent Vials,1-9
Tray Configuration,1-11
Inlet Buffer Tray,1-11
Outlet Buffer Tray,1-12

A51970AC
xii
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
Detector Initial Conditions (All Methods),2-2
Capillary Conditioning Method (SDS MW Conditioning - PA 800
plus.met),2-2
SDS-MW Separation Method,2-3
Shutdown Method,2-3

A51970AD 1-1
CHAPTER 1
SDS-MW Analysis Kit
Introduction
Capillary electrophoresis (CE) has become an effective replacement for manual slab gel
electrophoresis processes due to its automation, quantitation, fast speed, and high efficiency. Many
biomolecules, such as proteins, carbohydrates, and nucleic acids are separated by molecular sieving
electrophoresis using gel matrices, a technique referred to as capillary gel electrophoresis (CGE).
The separation results from the differential migration of the analyte through a gel matrix. In this
case, smaller molecules will move faster than large molecules through the separation gel. For
polypeptides and proteins, it is necessary to denature the sample in the presence of SDS, an anionic
detergent that binds the proteins in a constant ratio of 1:1.4 of protein. The constant mass-to-
charge property of the SDS-bound proteins allows separation according to differences in protein
molecular weight.
The SDS-MW Analysis Kit is designed for the separation of protein-SDS complexes using a
replaceable gel matrix. The gel is formulated to provide an effective sieving range of approximately
10 kDa to 225 kDa. Within this size range, the logarithm of protein molecular mass is linear with its
reciprocal electrophoretic mobility. Therefore, the molecular weight of an unknown protein may
be estimated from a standard curve of known protein sizes. This kit can also be used to quantify the
amount of protein and to determine the purity of a protein product.
NOTE 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. The SDS-MW Size
Standard is used to calibrate the gel to estimate the protein molecular weight of your sample. It also
provides confirmation of the resolving power of your experiment.
Internal Standard
A 10 kDa protein is used as a mobility marker. The mobility of all protein samples are calculated
relative to this mobility marker allowing for more accurate size estimation and analyte
identification.

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1-2
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 Quantity
Capillary, 50 µm I.D. bare-fused silica 2
SDS-MW Gel Buffer - proprietary formulation, pH 8, 0.2% SDS 140 mL
SDS-MW Sample Buffer - 100 mM Tris-HCl pH 9.0, 1%SDS 50 mL
SDS-MW Size Standard (10 to 225 kDa, 16 mg/mL) 100 µL
Internal Standard, 10 kDa, protein 5mg/mL 0.4 mL
Acidic Wash Solution (0.1 N HCl) 100 mL
Basic Wash Solution (0.1 N NaOH) 100 mL
Table 1.2 Replacement Reagents
Component Quantity Part Number
Capillary, 50 µm I.D. bare-fused silica pkg of 3 338451
SDS-MW Gel Buffer - proprietary formulation, pH 8, 0.2% SDS pkg of 4 A30341
SDS-MW Size Standard (10 to 225 kDa, 16 mg/mL) pkg of 3 A22196
Internal Standard, 10 kDa protein, 5mg/mL pkg of 1 A26487

A51970AD 1-3
SDS-MW Analysis Kit
Storing the Assay Components 1
Storing the Assay Components
Reagents
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
Beckman Coulter Consumables
Component Part Number
2-mercaptoethanol Sigma-Aldrich, PN M7154,
M6250
Water Bath (37°C to 100°C) or Heat Block
Centricon YM-10 Centrifugal Filter Unit Millipore, PN 4205
Iodoacetamide Sigma-Aldrich, PN I-1149
Parafilm
Sonicator
Vortexer
Pipets of various sizes with corresponding pipet tips
Micro-centrifuge
Component Part Number
0.5mL Micro-centrifuge Capped Vials (pack of 500) 344319
1.5 mL Centrifuge Tubes (pack of 500) 357448
Universal Plastic Vials (pack of 100) A62251
Universal Rubber Vial Caps - blue (pack of 100)a
a. Vial caps are one time use only and are not intended for re-use.
A62250
200 µL Micro Vials (pack of 50) 144709
Double-deionized (DDI) water

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1-4
SDS-MW Analysis Kit
Preparing the PA 800 plus Instrument
Preparing the PA 800 plus Instrument
NOTE Before proceeding, you must understand the following procedures as described in the PA 800 plus
System Maintenance Guide (PN A51964AD or the most current version):
• Capillary Replacement
• Installation of the PDA detector
• How to calibrate the PDA detector
• How to load and unload trays
Installing the Capillary
1 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.
2 Use the 100 x 200 µm capillary aperture for this installation.
To get good reproducibility from capillary to capillary and accurate mobility assignments, it is
important to adhere to the capillary pre-measurement procedure.
NOTE The cut ends of capillaries should be inspected carefully under magnification. The cut must be
clean (not jagged) and perpendicular to the capillary length (not angled). Poor cuts will result in poor
resolution and poor sample loading.
3 Turn off the PA 800 plus instrument and install the PDA detection module.
4 Turn on the instrument and permit the UV lamp to warm up for at least 30 minutes prior to
experimentation.
Cleaning the Interface
Following System Maintenance Guide A51964AD or latest revision, carefully clean the electrodes,
capillary ends, and interface block either every other day or after the finish of the sequence. The
SDS gel buffer is very viscous and will accumulate on the electrodes, capillary ends, opening levers,
and interface block if regular and thorough cleaning is not employed. Gel accumulation may cause
various modes of system failure, including broken capillaries and bent electrodes, vial jams, and
missed injections.

A51970AD 1-5
SDS-MW Analysis Kit
Sample Preparation 1
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
1 Remove SDS-MW Size Standard from refrigerator. Leave at room temperature for 15 minutes
before starting sample preparation.
2 Mix the standard thoroughly and centrifuge briefly in a standard microfuge.
3 Pipette 10 µL of Size Standard into a micro-centrifuge vial.
4 Add 85 µL of Sample Buffer into the micro-centrifuge vial.
5 Add 2 µL of Internal Standard into the micro-centrifuge vial.
6 Add 5 µL of 2-mercaptoethanol. Cap the vial tightly, seal with Parafilm, and mix thoroughly.
7 Heat mixture in a water bath at 100°C for three minutes.
8 Place the vial in a room-temperature water bath to cool for five minutes before injection. The
sample will remain stable for approximately 24 hours.

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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. The sample should be desalted with a Centricon column using the following procedure:
1 Add 1 mL of protein sample to a Centricon YM-10, then add 1 mL SDS-MW sample buffer into
the Centricon.
2 Centrifuge at 7,000 g for 20 minutes.
3 Add 2 mL of sample buffer. Centrifuge at 7,000 g for 20 minutes.
4 Insert the Centricon upside-down to drain the suspended protein solution (in the filter
membrane) into a new vial and centrifuge for three minutes at 5,000 g.
5 Transfer the collected protein to an appropriate sterile tube. Add sample buffer to give a final
volume of 1 mL.
Protein Sample Concentration
After the addition of the SDS-MW Sample Buffer, the total protein concentration should be within
the range of 0.2 mg/mL to 2 mg/mL. For best results, the recommended protein concentration is
1 mg/mL. If the protein concentration is too high, it can result in insufficient SDS binding, giving
broad peaks and poor resolution. On the other hand, if the protein concentration is too low, a low
signal will be observed.
Reducing the Protein Sample
Reduction of the disulfide bonds will provide a more accurate assessment of the molecular weight
of a protein, and will allow you to gain additional structural information on a given protein. In this
case:
1 Dilute the sample with the SDS-MW Sample buffer for a total 95 µL volume to give a final protein
concentration range of 0.2 mg/mL to 2 mg/mL.
2 Add 2 µL of Internal Standard.

A51970AD 1-7
SDS-MW Analysis Kit
Sample Preparation 1
3 Add 5 µL of 2-mercaptoethanol. Cap the vial tightly, seal with Parafilm, and mix thoroughly.
4 Heat mixture in a water bath at 100°C for 3 minutes.
5 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. In this case, it is a good idea to alkylate your protein sample to minimize any
heterogeneity created from partial auto-reduction of your protein. The following procedure is
recommended.
Preparing Alkylation Reagent
A 250 mM solution of iodoacetamide (IAM) is recommended as an alkylation reagent. The solution
is stable for approximately 24 hours at room temperature.
1 Weigh out 46 mg of IAM and place in a micro-centrifuge vial.
2 Add 1 mL of DDI water to a sample vial. Cap the vial tightly and mix thoroughly.
Preparing the Sample
1 Dilute the sample with the SDS-MW Sample Buffer for a total 95 µL volume to give a final
protein concentration range of 0.2 mg/mL to 2 mg/mL.
2 Add 2 µL of Internal Standard.
3 Add 5 µL of IAM solution.
4 Cap the vial tightly, seal with Parafilm, and mix thoroughly.
5 Heat mixture in a water bath at 70°C for 3 minutes.

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SDS-MW Analysis Kit
Sample Vial Setup
6 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. If bubbles exist, centrifuge the micro vials for 2
minutes at 1,000 g and repeat if necessary. Place a blue cap on the universal vial and ensure a good
seal. See Figure 1.1.
Place the universal vials into the 48-position inlet sample tray from positions A1 through C8.
Figure 1.1 Sample Vial Setup
1. Universal Cap
2. Micro Vial
3. Universal Vial
4. Micro Vial inside Universal Vial