Application Guide
PA 800 plus
Pharmaceutical Analysis
System
Carbohydrate Labeling and Analysis
A51969AA
April 2009
Beckman Coulter, Inc.
4300 N. Harbor Blvd.
Fullerton, CA 92835
Application Guide
PA 800 plus Pharmaceutical Analysis System
Carbohydrate Labeling and Analysis
A51969AA (April 2009)
Copyright © 2009 Beckman Coulter, Inc.
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.
Trademarks:
Following is a list of Beckman Coulter trademarks:
• Beckman Coulter®
• 32 Karat™
All other trademarks are the property of their respective
owners.
Find us on the World Wide Web at:
www.beckmancoulter.com and www.CELeader.com
Beckman Coulter Ireland Inc.
Mervue Business Park,
Mervue, Galway,
Ireland (353 91 774068)
Beckman Coulter do Brasil Com e Imp de Prod de Lab Ltda
Estr dos Romeiros, 220 - Galpao G3 - Km 38.5
zip code 06501-001 - Sao Paulo - SP - Brasil
CNPJ: 42.160.812/0001-44
製造販売元 : ベ ッ ク マ ン･ コ ール タ ー株式会社
東京都江東区有明二丁目 5 番 7 号
生产商：
贝克曼库尔特有限公司，美国加利福尼亚州富勒顿市，
邮编：92835，电话：(001）714-871-4848
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
A51969AA
iii
Revision History
iv
A51969AA
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.
A51969AA
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 “THIS PRODUCT CONFORMS TO APPLICABLE REQUIREMENTS OF 21 CFR 1040 AT THE
DATE OF MANUFACTURE” is found near the Name Rating tag. The laser light beam is not visible.
CLASS 1 LASER PRODUCT
THIS PRODUCT CONFORMS TO
APPLICABLE REQUIREMENTS OF
21 CFR 1040 AT THE DATE OF
MANUFACTURE.
MANUFACTURED:
726024-C
A016350L.EPS
Sharp Object Label
A label reading “CAUTION SHARP OBJECTS” is found on the PA 800 plus.
CAUTION
SHARP OBJECTS
A16558-AA
A016351L.EPS
vi
A51969AA
Safety Notices
Symbols and Labels
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.
A51969AA
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.
RoHS Caution Label
This logo indicates that this electronic information product contains certain toxic or hazardous
elements, and can be used safely during its environmental protection use period. The number in the
middle of the logo indicates the environmental protection use period for the product. The outer
circle indicates that the product can be recycled. The logo also signifies that the product should be
recycled immediately after its environmental protection use period has expired. The date on the
label indicates the date of manufacture.
RoHS Environmental Label
This logo indicates that the product does not contain any toxic or hazardous substances or
elements. The "e" stands for electrical, electronic and environmental electronic information
products. This logo indicates that this electronic information product does not contain any toxic or
hazardous substances or elements, and is green and is environmental. The outer circle indicates
that the product can be recycled. The logo also signifies that the product can be recycled after being
discarded, and should not be casually discarded.
viii
A51969AA
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.
A51969AA
ix
Safety Notices
Symbols and Labels
x
A51969AA
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, vi
Recycling Label, vii
Disposal of Devices Containing Mercury Components, vii
Restriction of Hazardous Substances (RoHS) Labels, viii
RoHS Caution Label, viii
RoHS Environmental Label, viii
Alerts for Warning, Caution, Important, and Note, ix
Carbohydrate Labeling and Analysis Kit, 1
Overview, 1
Safety Information, 1
General Precautions, 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, 3
Enzymatic Release of the N-Linked Oligosaccharides, 4
Working with the Supernatant, 4
Principle of the Labeling Method, 4
Preparing the Labeling Reagents, 5
Choosing the Proper Fluorophore, 5
xi
Contents
Preparing the Reagents, 5
Preparing the Standards, 6
Performing the Labeling Reaction, 6
Preparing the PA 800 Instrument, 8
Installing and Pre-conditioning the N-CHO Capillary, 9
Clean the Interface Block, 10
Insert the Cartridge, 10
Preparing the Buffer Trays, 10
Sample Vial Setup, 12
Running the Samples, 13
Initial Conditions, 13
LIF Detector Initial Conditions, 14
Time Program, 15
Evaluation of Results, 17
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, 21
CCF Troubleshooting Procedures, 24
CCF <1, 24
CCF >1, 24
No Step Change Detected, 24
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 The Carbohydrate Labeling and Analysis Kit is for laboratory use only.
IMPORTANT In this procedure use 1 M sodium cyanoborohydride in THF, which is a very reactive compound.
Please consult the MSDS before using or disposing of this reagent.
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.
CAUTION
When installing or removing capillaries from the cartridge, use safety goggles.
A51969AA
1
Carbohydrate Labeling and Analysis Kit
Materials and Reagents
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).
• 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.
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
Glucose Ladder Standard
50 mg
G20
0.18 mg
G22
Carbohydrate Separation Buffer
N-CHO Coated Capillary
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
Spatula
Analytical scale
Vortexer
2
A51969AA
Carbohydrate Labeling and Analysis Kit
Overview of the Procedure
Description
Part Number
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.
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):
A51969AA
3
Carbohydrate Labeling and Analysis Kit
Releasing Oligosaccharides From Glycoproteins
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, and 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.
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
8-aminopyrene-1,3,6=, 6-trisulfonate. 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.
4
A51969AA
Carbohydrate Labeling and Analysis Kit
Preparing the Labeling Reagents
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.
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.
A51969AA
5
Carbohydrate Labeling and Analysis Kit
Performing the Labeling Reaction
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.
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.
6
A51969AA
Carbohydrate Labeling and Analysis Kit
Performing the Labeling Reaction
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 welland transfer the solution into a micro vial for analysis in the PA 800
system see Sample Vial Setup.
Figure 3 illustrates a summary of the sample preparation for injection.
Figure 2 Labeling Reaction Scheme
A51969AA
7
Carbohydrate Labeling and Analysis Kit
Preparing the PA 800 Instrument
Figure 3 Sample Preparation for Injection
Preparing the PA 800 Instrument
Before proceeding, review the following procedures in the PA 800 plus System Maintenance User’s
Guide (A51964AA).
• Install the LIF Detector
• Calibrate the LIF Detector
• Capillary Cartridge Procedures
• Cleaning the Interface Block and the Opening Levers
8
A51969AA
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 (A51964AA).
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 system and then install the LIF detection components.
• Turn on the system and permit the laser to warm up for at least 30 minutes, prior to calibration.
A51969AA
9
Carbohydrate Labeling and Analysis Kit
Preparing the Buffer Trays
Clean the Interface Block
Clean the electrodes, opening levers, capillary tips, and interface block carefully using a Kimwipe
soaked with DDI water. Do this on a weekly basis or when changing chemistries on the PA 800
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
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)
10
A51969AA
Carbohydrate Labeling and Analysis Kit
Preparing the Buffer Trays
Figure 5 Universal Vials and Caps
1. Universal Vial Cap
2. Maximum Fill Level
3. Universal Vials
2
Cover each vial with a cap as shown in Figure 5. Caps are not intended for re-use and should be
disposed of after the buffer used is exhausted. Re-using vial caps can result in pressure leakage
and capillary breakage.
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.
A51969AA
11
Carbohydrate Labeling and Analysis Kit
Sample Vial Setup
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. For the best
quantitative results, perform one injection per vial, introducing replicates in separate vials.
12
A51969AA
Carbohydrate Labeling and Analysis Kit
Running the Samples
Figure 7 Micro Vial Inside a Universal Vial
1. Universal Cap
2. Micro Vial
3. Universal Vial
4. Micro Vial inside Universal Vial
Running the Samples
Initial Conditions
Table 2 Test Run: Initial Conditions
A51969AA
Capillary Temperature:
20°
Sample Storage Temperature:
10°
Auxiliary Data Channel:
Current
13
Carbohydrate Labeling and Analysis Kit
Running the Samples
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
A51969AA
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.
A51969AA
15
Carbohydrate Labeling and Analysis Kit
Running the Samples
3
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.
4
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
A51969AA
Carbohydrate Labeling and Analysis Kit
Evaluation of Results
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.
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.
A51969AA
17
Carbohydrate Labeling and Analysis Kit
System Shutdown and Capillary Storage
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
18
1
Perform a 3 minute, 30 psi rinse with water and then with Carbohydrate Separation Buffer for
3 minutes.
2
Remove the capillary from the instrument and place in a cassette box with the capillary ends
placed in vials of DDI water.
3
Store the cartridge box at 2°C to 8°C in an upright position.
A51969AA
Carbohydrate Labeling and Analysis Kit
Troubleshooting Procedures
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 indicating
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 and 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.
A51969AA
Low Current
• Use approximately 50 cm N-CHO capillary total length (50 μm I.D.) for a
predicted current that should be steady at around -15 μA. If the current
is lower, the capillary may be blocked. Use Direct Control to perform a
70 psi rinse with water to unblock the capillary.
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.
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.
19
Carbohydrate Labeling and Analysis Kit
Appendix
Appendix
LIF Detector Calibration Procedure
The PA 800 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.
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.
4
Select the CHO instrument icon and then Configure Instrument.
5
Select Configure on the instrument configuration window.
6
Select the LIF Detector icon in the right pane of the PA 800 System Configuration window.
7
The 32 Karat Software Configuration dialog box appears as shown in Figure 13.
A51969AA
Carbohydrate Labeling and Analysis Kit
Appendix
Figure 13 32 Karat Software Configuration dialog
8
Select LIF Calibration Wizard from the Instrument Configuration window to display the
Calibration Wizard. See Figure 14.
Automatic Calibration
1
A51969AA
Choose Auto as the calibration option, as shown in Figure 14 and click Next.
21
Carbohydrate Labeling and Analysis Kit
Appendix
Figure 14 Calibration Wizard Screen - Auto Select
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
22
A51969AA
Carbohydrate Labeling and Analysis Kit
Appendix
3
Fill one buffer vial with 1.5 mL DDI water and another with 200 μL of diluted LIF Performance
Test Mix (1:1 with DDI water) in a micro vial. Place them in the buffer vial positions as indicated
in Figure 16.
4
Place an empty vial in the waste position.
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.
A51969AA
23
Carbohydrate Labeling and Analysis Kit
Appendix
Figure 17 Calibration Wizard Screen - Step 4
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 that the laser provided with the PA 800 was used.
• 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 that the laser provided with the PA 800 is being used for
calibration.
• 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 one rinses with the non-fluorescent solution
24
A51969AA
Carbohydrate Labeling and Analysis Kit
Appendix
followed by a fluorescent solution rinse, one 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
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.
2
Once the rinse has begun, lift the sample cover.
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 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.
A51969AA
25
www.beckmancoulter.com and
www.CELeader.com
© 2009 Beckman Coulter, Inc.
All Rights Reserved