Application Guide
PA 800 plus
Pharmaceutical Analysis
System
Carbohydrate Labeling and Analysis
A51969AD
January 2014
Beckman Coulter, Inc.
250 S. Kraemer Blvd.
Brea, CA 92821 U.S.A.
Application Guide
PA 800 plus Pharmaceutical Analysis System
Carbohydrate Labeling and Analysis
A51969AD (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. and are registered in the USPTO.
No part of this document may be reproduced or
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Revision History
Initial Issue, A51969AA, April 2009
32 Karat Software version 9.1
PA 800 plus Software version 1.1
PA 800 plus Firmware version 9.0
First Revision, A51969AB, December 2009
Revised corporate address.
Second Revision, A51969AC, 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, A51969AD, January 2014
Formatting updates, Aminopyrene Trisulfonic Acid naming standardization.
A51969AD
iii
Revision History
iv
A51969AD
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.
A51969AD
v
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.
144557-AB
A016352L.EPS
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.
vi
A51969AD
Safety Notices
Symbols and Labels
Sharp Object Label
A label reading “CAUTION SHARP OBJECTS” is found on the PA 800 plus.
CAUTION
SHARP OBJECTS
A16558-AA
A016351L.EPS
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.
A51969AD
vii
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.
viii
A51969AD
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.
A51969AD
ix
Safety Notices
Symbols and Labels
x
A51969AD
Contents
Revision History, iii
Safety Notices, v
Symbols and Labels, v
Introduction, v
General Biohazard Symbol, v
Caution, Biohazard Label, v
Caution, Moving Parts Label, vi
High Voltage Electric Shock Risk Symbol, vi
Class 1 Laser Caution Label, vi
Sharp Object Label, vii
Recycling Label, vii
Disposal of Devices Containing Mercury Components, vii
Restriction of Hazardous Substances (RoHS) Labels, viii
Alerts for Warning, Caution, Important, and Note, ix
Carbohydrate Labeling and Analysis Kit, 1
Overview, 1
Safety Information, 1
General Precautions, 1
Intended Use, 2
Materials and Reagents, 2
Contents of this Kit, 2
Materials Needed but Not Provided in Kit, 2
Storage Conditions, 3
Overview of the Procedure, 3
Releasing Oligosaccharides From Glycoproteins, 4
Enzymatic Release of the N-Linked Oligosaccharides, 4
Working with the Supernatant, 4
Principle of the Labeling Method, 5
Preparing the Labeling Reagents, 5
Choosing the Proper Fluorophore, 5
Preparing the Reagents, 6
Preparing the Standards, 6
xi
Contents
Performing the Labeling Reaction, 7
Preparing the PA 800 plus Instrument, 9
Installing and Pre-conditioning the N-CHO Capillary, 10
Clean the Interface Block, 11
Insert the Cartridge, 11
Preparing the Buffer Trays, 11
Sample Vial Setup, 12
Running the Samples, 14
Initial Conditions, 14
LIF Detector Initial Conditions, 14
Time Program, 15
Evaluation of Results, 18
System Shutdown and Capillary Storage, 18
Short Term Storage (<24 hours) of Capillary, 18
Long Term Storage (>24 hours) of Capillary, 18
Troubleshooting Procedures, 19
Appendix, 20
LIF Detector Calibration Procedure, 20
Setting the Calibration Corrector Factors (CCFs), 20
Automatic Calibration, 22
CCF Troubleshooting Procedures, 25
CCF <1, 25
CCF >1, 25
No Step Change Detected, 25
xii
Carbohydrate Labeling and Analysis Kit
Overview
Beckman Coulter’s Carbohydrate Labeling & Analysis Kit uses capillary electrophoresis to separate
and quantify oligosaccharides released from glycoproteins. This kit has been developed to work
with the PA 800 plus Pharmaceutical Analysis System, using the 488 nm laser and the 520 nm
emission filter.
This kit contains the reagents, buffer, and coated capillaries required to label, separate, and
quantify oligosaccharides. This kit also provides a glucose size marker for relative size
determination and a maltose standard for quantitation and mobility characterization of the
released oligosaccharides.
For further information, please refer to the following publication:
Quantitative Analysis of Sugar Constituents of Glycoproteins by Capillary Electrophoresis, Fu Tai, A.
Chen, Thomas S. Dobashi and Ramon A. Evangelista, “Glycobiology”, Vol. 8, No. 11, pp. 1045-1052,
1998.
Safety Information
NOTE This application guide has been validated for use in the PA 800 Enhanced Protein Characterization and
the PA 800 plus Pharmaceutical Analysis Systems.
IMPORTANT In this procedure 1 M sodium cyanoborohydride in THF is used, which is a very reactive
compound. Please consult the MSDS before using or disposing of this reagent.
CAUTION
When installing or removing capillaries from the cartridge, use safety goggles.
General Precautions
• Prior to using the instrument, turn on the laser and let it stabilize for 15 minutes. Turn it off
when not in use for longer periods of time.
• Do not interchange the capillary (i.e. if a capillary is being used for carbohydrate analysis, do
not use the same capillary in another application).
A51969AD
1
Carbohydrate Labeling and Analysis Kit
Materials and Reagents
• When the capillary is not in use or after finishing with all experiments, it is recommended to
run a shutdown method to rinse the capillary and leave the ends immersed in water to avoid
capillary blockage and to keep the capillary from drying out.
• Prior to using the capillary for the first time, it is recommended to rinse the capillary for 10
minutes at 30 psi pressure with DDI water and then rinse for 10 minutes at 30 psi with the
Carbohydrate Separation Gel Buffer.
Intended Use
The Carbohydrate Labeling and 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
Contents of this Kit
Table 1 Kit Contents Reorder Number (477600)
Component
Quantity
Reorder #
56 mL
477623
2
477601
Labeling Dye (APTS)
4x5 mg
501309
Labeling Dye Solvent
1 mL
L3
50 mg
G20
0.18 mg
G22
Carbohydrate Separation Buffer
N-CHO Coated Capillary
Glucose Ladder Standard
Quantitation/Mobility Marker (Maltose)
APTS-M (monosaccharide grade)
20 mg
Code
L6
725898
Materials Needed but Not Provided in Kit
Description
Part Number
LIF Performance Test Mix
726022
Universal Plastic Vials (pack 100)
A62251
Universal Rubber Vial Caps - blue (pack 100)
A62250
200 µL Micro Vials (pack 50)
144709
Assorted pipettors and pipette tips
2
A51969AD
Carbohydrate Labeling and Analysis Kit
Overview of the Procedure
Description
Part Number
Spatula
Analytical scale
Vortexer
Bench top micro-centrifuge
Water bath
Centrifugal vacuum evaporator
Sodium Cyanoborohydride, 1 M/THF
Sigma-Aldrich
296813
2-mercaptoethanol
Sigma-Aldrich
M7154
Double Deionized (DDI) water filtered through 0.2 µm filter
N-glycosidase F (PNGase F)
New England
Biolabs P0704S
Nonidet® NP-40 non ionic detergent
Storage Conditions
The kit components are stable for one year when stored under the following conditions:
• Upon receipt, the kit should be stored at 2 to 8°C.
• Reconstituted Quantitation Control (G22) and labeled glucose ladder standard (G20) should be
stored at -35 to -15°C.
• Reconstituted labeling reagent (APTS) shall be stored at -35 to -15°C. It is stable for 2 weeks after
reconstitution.
Overview of the Procedure
The carbohydrate analysis involves the following three tasks:
• Enzymatic release of the N-linked oligosaccharides from the glycoprotein.
• Labeling of the mixture of released oligosaccharides.
• Analysis of the flouorophore-labeled oligosaccharides by capillary electrophoresis.
A51969AD
3
Carbohydrate Labeling and Analysis Kit
Releasing Oligosaccharides From Glycoproteins
Releasing Oligosaccharides From Glycoproteins
IMPORTANT This labeling kit does not contain releasing enzymes.
There are multiple enzymatic and chemical procedures for releasing oligosaccharides from
proteins. In order to successfully label the released oligosaccharide, it is essential that the reducing
termini of the oligosaccharide is not destroyed by the deglycosylation method.
The following is a suggested protocol for N-deglycosylation, using N-Glycosidase F (PNGase F):
Enzymatic Release of the N-Linked Oligosaccharides
1. Aliquot the sample volume in order to have between 25-300 µg of glycoprotein.
2. Dry the glycoprotein solution completely in a centrifugal vacuum evaporator.
3. Add 45 µL of 1X PBS buffer.
4. Add 1.0 µL of 5% SDS (or a volume that gives final concentration of 0.1% SDS).
5. Add 1.5 µL of 1:10 dilution of 2-mercaptoethanol in DDI water (or a volume that gives final
concentration of 50 mM 2-mercaptoethanol).
6. Heat the sample for 5 minutes at 100°C.
IMPORTANT If the denatured protein precipitates, discard the sample and restart this process, repeating
steps 1 through 5. For step 6, denature the protein by incubating it at 37°C for 10 minutes.
7. Cool the sample to room temperature.
8. Add 5 µL of Nonidet® NP40 non-ionic detergent (or a sufficient volume that gives a final
concentration of 0.75%).
9. Add PNGase F enzyme, according to its activity.
10. React for 15 hours at 37°C.
11. Add approximately 150 µL of cold ethanol (or 3 times the actual reaction mixture volume).
12. Vortex the mixture and place it on ice for complete protein precipitation.
13. Centrifuge the samples at 15,000 rpm for 5 minutes.
14. Withdraw and save the supernatant.
IMPORTANT This solution contains the RELEASED N-LINKED OLIGOSACCHARIDES.
15. Discard the solid. This is the deglycosylated protein.
Working with the Supernatant
There are two options for working with the supernatant:
For quantitative analysis, add an internal standard to the supernatant. See Preparing the Standards
beginning with Step 1a.
4
A51969AD
Carbohydrate Labeling and Analysis Kit
Preparing the Labeling Reagents
For qualitative analysis, bring the supernatant to dryness in the centrifugal vacuum evaporator.
These oligosaccharides are ready to be labeled with APTS. See Performing the Labeling Reaction in
this chapter.
Principle of the Labeling Method
After release (enzymatic or chemical), the oligosaccharides can be labeled with a fluorophore called
1-Aminopyrene-3,6,8 – Trisulfonic Acid (APTS). The stoichiometry of labeling reaction is one APTS
molecule per molecule of oligosaccharide. Figure 1 illustrates the labeling reaction of an N-linked
oligosaccharide with APTS.
Figure 1 Labeling Reaction of an Oligosaccharide with APTS
The efficiency of the labeling reaction is dependent on temperature and the amount of
oligosaccharides. This protocol has been optimized for labeling 5 nmoles or less of total
oligosaccharides.
Samples with amounts higher than 5 nmoles may give a lower reaction yield. Use maltose (G22) as
an internal labeling control and/or as an internal mobility marker.
Preparing the Labeling Reagents
Choosing the Proper Fluorophore
APTS-M is a high-purity fluorophore intended for labeling monosaccharides. APTS-M contains
citric acid as a catalyst. APTS (L6) can be used for oligosaccharides.
A51969AD
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Carbohydrate Labeling and Analysis Kit
Preparing the Labeling Reagents
Preparing the Reagents
1
Prepare the APTS labeling reagent.
To a vial of APTS labeling dye (L6)
a. Add 48 µL of Labeling dye solvent (15% acetic acid – L3).
b. Vortex for 5 seconds until complete dissolution.
c. Store at -35 to -15°C for up to 2 weeks when not in use.
2
Prepare the APTS-M (Monosaccharide grade) Labeling Dye
a. Add 400 µL of DDI water to the APTS-M vial.
b. Vortex the mixture for 5 seconds until all of the solid is dissolved.
c. Store the reconstituted APTS-M at -35 to -15°C for up to two weeks.
Preparing the Standards
1
Prepare the Maltose Quantitative Control/Mobility Marker (G22).
a. Add 500 µL of DDI water to the Maltose quantitative control labeled G22. This makes a
solution containing 500 nmoles of maltose, or concentration of 1 nmol/µL.
b. For quantitation purposes, add 5 µL of G22 reconstituted to your released oligosaccharides.
c. Dry down the sample in a centrifugal evaporator and proceed to labeling protocol, as
described in the next section.
d. Store at -35 to -15°C when not in use.
NOTE When using the maltose solution for quantitation purposes, make sure that the solution is
prepared fresh and used right away. Bacterial contamination can digest the sugars, leading to a
miscalculation of your quantitation.
2
Prepare the Glucose Ladder Standard (G20).
a. Weigh and dissolve 5 mg of Glucose Ladder Standard (G20) in 80 µL DDI water in 1.5 mL
microcentrifuge tube. Sonicate if necessary.
b. Aliquote at least ten 2 µL portions of the Glucose ladder standard solution to 0.5 mL
microcentrifuge vials and dry them in a centrifugal vacuum evaporator. The dried glucose
ladder can be stored at room temperature or used immediately.
6
A51969AD
Carbohydrate Labeling and Analysis Kit
Performing the Labeling Reaction
Performing the Labeling Reaction
1
Label the Sample and/or Standard (G20).
a. Add 2 µL of 1 M sodium cyanoborohydride/THF to the dried oligosaccharide sample.
b. Add 2 µL of APTS Labeling Reagent to the sample.
IMPORTANT Due to the reaction of sodium cyanoborohydride with water, avoid moisture and
store this product under dry conditions. Use a dry needle to dispense this chemical. Pass dry
argon into the chemical bottle through the septum while dispensing 2 µL from the bottle.
c. Incubate the reaction mixture at 37°C for 4 hours, or at 60°C for 90 minutes, or at room
temperature overnight (mildest reaction).
NOTE A mild labeling condition is necessary to avoid losing the sialic acid residues from your
oligosaccharide chain. You can leave the reaction mixture at room temperature overnight and in the
dark to do this. For Glucose Ladder incubation, select any of the temperature/ time options.
Figure 2 illustrates a summary of the overall labeling protocol.
2
Prepare the Sample and/or Standard for capillary electrophoresis.
a. To the 4 µL reaction mixture prepared above add:
• For Glucose Ladder Standard: add 96 µL of DDI water
• For all other samples: add 46 µL DDI water
This solution can be stored at 2 to 8°C.
b. Take an aliquot of 5 µL of the solution above and add 195 µL of DDI water.
c. Mix the contents well and transfer the solution into a micro vial for analysis in the PA 800
plus system see Sample Vial Setup.
Figure 3 illustrates a summary of the sample preparation for injection.
A51969AD
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Carbohydrate Labeling and Analysis Kit
Performing the Labeling Reaction
Figure 2 Labeling Reaction Scheme
8
A51969AD
Carbohydrate Labeling and Analysis Kit
Preparing the PA 800 plus Instrument
Figure 3 Sample Preparation for Injection
Preparing the PA 800 plus Instrument
Before proceeding, review the following procedures in the PA 800 plus System Maintenance User’s
Guide (A51964AD).
• Install the LIF Detector
• Calibrate the LIF Detector
• Capillary Cartridge Procedures
• Cleaning the Interface Block and the Opening Levers
A51969AD
9
Carbohydrate Labeling and Analysis Kit
Installing and Pre-conditioning the N-CHO Capillary
Installing and Pre-conditioning the N-CHO Capillary
• Prior to using the PA 800 plus system for carbohydrate analysis, the N-CHO capillary must be
installed in a capillary cartridge. To properly install the capillary, refer to the Capillary
Cartridge sections in the PA 800 plus System Maintenance User’s Guide (A51964AD).
IMPORTANT Be sure to pre-rinse a new capillary for 10 minutes at 30 psi pressure with DDI water and then
rinse for 10 minutes at 30 psi with Carbohydrate Separation Buffer prior to the first run.
For this analysis, you will be using a 50 µm I.D. N-CHO capillary. The length from injection inlet to
detector should be 40 cm, with the total length being 50.2 cm. Be sure to measure the capillary
dimensions accurately and record the dimensions in the capillary/performance tab of the
Advanced Method Options. See Figure 4. This will be necessary for accurate mobility
determinations.
Figure 4 Capillary Performance Screen
IMPORTANT To get good reproducibility from capillary to capillary and accurate mobility assignments, it is
important to adhere to the capillary pre-measurement procedure.
IMPORTANT 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.
NOTE When not in use, submerge the capillary ends in DDI water to prevent the capillary from drying out.
• Turn off the PA 800 plus system and install the LIF detection components.
• Turn on the system and permit the laser to warm up for at least 30 minutes prior to calibration.
10
A51969AD
Carbohydrate Labeling and Analysis Kit
Preparing the Buffer Trays
Clean the Interface Block
Clean the electrodes, opening levers, capillary tips, and interface block carefully following the
cleaning procedure as described in the PA 800 plus “System Maintenance” manual. Do this on a
weekly basis or when changing chemistries on the PA 800 plus system. Accumulation on the
interface block can lead to current leakage errors.
Insert the Cartridge
Insert the cartridge into the system. Close the cartridge and sample covers.
Preparing the Buffer Trays
1
Perform the LIF Detector Calibration Procedure in the Appendix.
2
Fill the vials with the appropriate volumes of each reagent:
• 1.5 mL of DDI water per H20 vial (4 vials)
• 1.5 mL of Carbohydrate Separation Buffer per Gel-R vial (1 vial)
• 1.3 mL of Carbohydrate Separation Buffer per Gel-S vial (2 vials)
• 0.8 mL of DDI water per waste vial (1 vial)
Figure 5 Universal Vials and Caps
1. Universal Vial Cap
2. Maximum Fill Level
3. Universal Vials
A51969AD
11
Carbohydrate Labeling and Analysis Kit
Sample Vial Setup
3
Place the reagent vials on the inlet and outlet buffer trays as indicated in Figure 6.
NOTE During the separation, salts migrate from one vial of gel buffer to the other, changing the
composition of the buffer. Therefore, it is recommended that the vials of buffer be replaced with fresh
buffer after 20 runs. For unattended operation of more than 20 runs, simply program the increment
function to increment the water and buffer vials after 20 runs. Vials will increment in a forward
direction. Ensure that the tray is filled with the appropriate number of vials to manage the number
of runs that are to be performed.
NOTE The H20 vials are used to clean the capillary tips after the sample introduction step to prevent
sample carryover. Replace the vials with fresh water if rinse water becomes contaminated.
A schematic of the buffer tray setup is illustrated in Figure 6.
Figure 6 Buffer Tray Configuration
Sample Vial Setup
Before placing the 200 microliter sample vials into the universal vials, ensure that no bubbles are at
the bottom of the sample vials. If bubbles exist, centrifuge the sample vials for two minutes at
1,000 g and repeat if necessary. Place a cap on the universal vial and ensure a good seal. See Figure 7.
Place the universal vials into the 48-position inlet sample tray positions A1 through C8.
12
A51969AD
Carbohydrate Labeling and Analysis Kit
Sample Vial Setup
Figure 7 Micro Vial Inside a Universal Vial
1. Universal Cap
2. Micro Vial
A51969AD
3. Universal Vial
4. Micro Vial inside Universal Vial
13
Carbohydrate Labeling and Analysis Kit
Running the Samples
Running the Samples
Initial Conditions
Table 2 Test Run: Initial Conditions
Capillary Temperature:
20°
Sample Storage Temperature:
10°
Auxiliary Data Channel:
Current
Figure 8 Initial Condition Tab
LIF Detector Initial Conditions
Table 3 Test Run: LIF Detector Initial Conditions
14
Detection
Laser Induced Fluorescence
Wavelength
Excitation - 488 nm, Emission - 520 nm
Data Rate
4 Hz
Dynamic Range
100 RFU (relative fluorescence units)
Filter Setting
Normal
Peak Width
16 - 25
A51969AD
Carbohydrate Labeling and Analysis Kit
Running the Samples
Figure 9 LIF Detector Initial Conditions Tab
NOTE Prior to use, calibrate the LIF detector using the procedure under LIF Detector Calibration Procedure.
For comparable results, the detection system should be calibrated at system start-up or whenever a
capillary is replaced.
Time Program
1
Rinse the capillary with buffer for three minutes at 30 psi from BI:B1 to waste at vial BO:B1.
2
Inject the sample at 0.5 psi for three seconds from sample vial to buffer vial BO:C1.
NOTE Either pressure or electrokinetic sample introduction can be used for labeled carbohydrate
samples. A 3 to 20 second 0.5 psi pressure injection is recommended. Longer injection times may
cause peak shape and migration times to vary. For low concentration samples, electrokinetic
injections may be the method of choice. The concentration and amount of sample will determine the
conditions of injection - typically 0.1 to 1.0 Ws (for example,10 kV, 5 microamps, 2-20 seconds)
should be used.
NOTE Salts will interfere with electrokinetic injections. Samples should first be desalted before
attempting this method.
A51969AD
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Carbohydrate Labeling and Analysis Kit
Running the Samples
3
4
Wait 0.2 minutes with vials BI:A4 and BO:A4. See Figure 10. This step dips the capillary in water
to protect against sample carry over. Change the rinse water vials if they are contaminated.
Separate Step - 20 minutes from vial BI:C1 to vial BO:C1 (buffer). See Figure 10. The constant
voltage should be at 30 kV, with reverse polarity and a 0.17 ramp time.
5
Autozero at 1.0 minute.
6
End at 20.0 minutes.
Figure 10 Time Program Tab
16
A51969AD
Carbohydrate Labeling and Analysis Kit
Running the Samples
Figure 11 Typical Current Profile
Field Strength Generated
598 V/cm
Typical Current. See Figure 11
under 20 µA
NOTE Prior to use, calibrate the LIF detector using the procedure under LIF Detector Calibration
Procedure. For comparable results, the detection system should be calibrated at system start-up or
whenever a capillary is replaced.
A51969AD
17
Carbohydrate Labeling and Analysis Kit
Evaluation of Results
Evaluation of Results
The test mixture contains the APTS-labeled glucose oligomers consisting of at least 20 individual
oligomers. An example of this test mix is highlighted in Figure 12.
Figure 12 Electropherogram - Glucose Ladder
System Shutdown and Capillary Storage
Short Term Storage (<24 hours) of Capillary
Perform a three minute, 30 psi rinse with water. The capillary may be stored on the instrument with
the capillary ends immersed in water.
Whenever the capillary has not been used for three hours or longer, rinse the capillary by
performing a three minute, 30 psi rinse with water before performing a separation.
Long Term Storage (>24 hours) of Capillary
1
18
Perform a 3 minute, 30 psi rinse with water. Repeat the rinse with Carbohydrate Separation
Buffer for 3 minutes.
A51969AD
Carbohydrate Labeling and Analysis Kit
Troubleshooting Procedures
2
3
Remove the capillary from the instrument and place in a cassette box with the capillary ends
placed in vials of DDI water.
Store the cartridge box at 2°C to 8°C in an upright position.
Whenever the capillary has not been used for three hours or longer, rinse the capillary by
performing a 3 minute, 30 psi rinse with water and then a 10 minute rinse with Carbohydrate
Separation Buffer before performing a separation.
Troubleshooting Procedures
Symptom
Corrective Action
No peaks
• Check if your LIF detector has been calibrated. If not, please refer to the
LIF calibration section of this system manual to perform the LIF
Calibration.
• Make sure that your laser is ON.
• Make sure the probe stabilizer is properly connected to the lock down
bar (white bar that locks down your LIF cartridge).
• Make sure that you set up voltage in the method properly. This
separation should be run using the reverse polarity. Check if the proper
polarity is being applied by confirming that the orange LED that
indicates reverse polarity is on during the separation.
• Make sure that there is no bubble trapped at the bottom of the sample
vial preventing the sample from being injected.
• Make sure the window of your capillary is intact. If the window is broken,
change the capillary right away. Most importantly, clean the probe
aligner with a cotton swab moistened in water.
Low Intensity Peaks
• Labeling reaction was not carried out properly. For a successful labeling
reaction, make sure that:
— the protein is in a non-tris buffer. Tris will compete with your
carbohydrate for the reductive amination.
— the carbohydrate pellets were fully dried. Any residue of water will
interfere in the yield of your labeling reaction.
• Enzyme activity may differ from lot to lot. You may need to adjust the
suggested amount, if the reaction gives a yield lower than what you
usually expect.
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Low Current
• Confirm N-CHO capillary with 50 cm length is in place. Confirm
temperature. Replace reagent vials with new reagent vials.
Shift in Migration time in
between runs, within the
same day
• Migration time shifts, with the use of this application, are associated with
the length of injection. Make sure that injections are between 3 - 20 s at
0.5 psi. The injection time depends on the concentration.
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Carbohydrate Labeling and Analysis Kit
Appendix
Symptom
Corrective Action
Carryover
• Change water dip vials or change buffer vials and increment these vials
as necessary
Spikes in Electropherogram
• Check for air that is dissolved in the gel buffer. Sonicate the gel for 5
minutes.
Extra Peaks
• It is very important to use extra clean 0.5 mL micro-centrifuge vials,
especially during the labeling step. Contaminants left over from the
manufacturing process of these vials may react with APTS, giving extra
peaks on separation.
Appendix
LIF Detector Calibration Procedure
The PA 800 plus LIF detector calibration kit procedure is used to calibrate the detection system for
carbohydrate analysis. This will ensure consistent results from day to day and from capillary to
capillary, as detection is measured in relative fluorescence units (RFU). This calibration procedure
allows you to normalize the signal generated from your analyte, relative to a known concentration
of sodium fluorescein. Perform the calibration procedure when installing a new capillary or after
detector and cartridge changes.
IMPORTANT The LIF calibration procedure described in this application guide has changed from previous
versions. The result is different RFU values than what were obtained from assays standardized on the
previous LIF calibration procedure. The calibration correction factor (CCF) is a scalar multiple of all data
points, so there is no impact on signal to noise performance. This change was made to reduce the risk of
saturating the detector from excessively high fluorescence signals.
Table 4 Reagents Needed to Perform the Procedure
Materials
LIF Performance Test Mix
DDI water
Part Number
726022
N/A
Setting the Calibration Corrector Factors (CCFs)
IMPORTANT The user must have instrument administration privileges.
20
1
Launch 32 Karat.
2
Go to Tools > Enterprise Login.
3
Enter the user name PA 800 and password plus and select Log in.
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Appendix
4
Select the CHO instrument icon and then Configure Instrument.
5
Select Configure on the instrument configuration dialog box.
6
Select the LIF Detector icon in the right pane of the PA 800 plus System Configuration dialog box.
7
The 32 Karat Software Configuration dialog box appears as shown in Figure 13.
Figure 13 32 Karat Software Configuration Dialog Box
8
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Select LIF Calibration Wizard from the Instrument Configuration dialog box to display the
Calibration Wizard. See Figure 14.
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Appendix
Automatic Calibration
1
Choose Auto as the calibration option, as shown in Figure 14 and click Next.
Figure 14 Calibration Wizard Screen - Auto Select
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2
Set up the target RFU value. The target value for N-CHO-coated capillary is 7, as shown in
Figure 15, and click Next.
Figure 15 Calibration Wizard Screen - Step 2
3
4
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Fill one buffer vial with 1.5 mL DDI water and another with 200 µL of diluted LIF Performance
Test Mix (1:2 with DDI water) in a micro vial. Place them in the buffer vial positions as indicated
in Figure 16.
Place an empty vial in the waste position.
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Carbohydrate Labeling and Analysis Kit
Appendix
IMPORTANT The system performance test kit (713360) contains instructions for performing detector noise
tests on a bare-fused silica capillary, which uses borate buffer. Do not use the borate buffer for calibration
of the N-CHO capillary. Use a 50% LIF Performance Test mix solution (100 µL of LIF Test Mix with 100 µL
of DDI water) in a micro vial.
5
Click Next to perform the automatic calibration, as shown below.
Figure 16 Calibration Wizard Screen - Step 3
6
After the calibration is completed, a dialog box displays as shown in Figure 17.
A number will show in the Calibration Correction Factor field.
If it is below 10, click Accept to complete the calibration procedure.
If it is above 10. See CCF Troubleshooting Procedures.
Figure 17 Calibration Wizard Screen - Step 4
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Appendix
CCF Troubleshooting Procedures
CCF <1
Table 5 CCF <1
Symptom
Corrective Action
CCF is still below 1, but not below 0.1
• There is no problem with the system. Run a standard and verify
acceptable performance of the system.
System performance is not acceptable
or the CCF is below 0.1
• Verify that the capillary for the carbohydrate analysis was
used, and it is not broken.
• Verify laser output for the laser in use on the PA 800 plus
system.
• Verify that the correct filters are installed in the LIF detector.
• Contact a Beckman Coulter service representative.
CCF >1
Table 6 CCF >1
Symptom
Corrective Action
CCF is more than 1.0 but less than 10
• Run a standard to determine if the system performance is still
acceptable.
CCF is more than 10 or if the system
performance is not acceptable.
• Verify laser output for the laser in use on the PA 800 plus
system.
• Verify that the correct filters are installed in the LIF detector.
• Replace the chemistries and capillary and repeat the
calibration.
• Contact a Beckman Coulter service representative.
No Step Change Detected
The LIF Calibration method is basically a comparison of detector signals between a non-fluorescent
solution to a known fluorescent solution. When rinsed with the non-fluorescent solution followed
by a fluorescent solution rinse, you should see the first part of the detector signal near zero and the
second part of the detector signal near the target fluorescent value. This detector output is step
shaped in appearance.
If no step change is observed, the appropriate solutions are not passing the detector or the detector
is not able to detect them.
1
2
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Verify that the solution is rinsing through the capillary by performing a direct control 5 minute
20 psi pressure rinse of water, from buffer inlet A1 to an empty buffer vial in outlet B1.
Once the rinse has begun, lift the sample cover.
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Carbohydrate Labeling and Analysis Kit
Appendix
3
Observe the outlet end of the capillary in B1. Droplets should be formed on the outlet end of the
capillary.
• If no droplets are formed, either the capillary is plugged or the instrument has a pressure
failure.
• Replace the capillary and repeat the rinse. If there is still no droplet observed, contact a
Beckman Coulter service representative.
Once flow through the capillary is verified, the detection system is the only other possible
cause, when no step is detected.
4
Verify that the correct filters are installed in the LIF detector.
5
Verify that the laser provided with the PA 800 plus system is connected and on.
6
Run the calibration test with a new capillary and chemistries.
If there is still no step change detected, contact a Beckman Coulter service representative.
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