n-linked oligosaccharide profiling manual

8/9/2019 N-Linked Oligosaccharide Profiling Manual

1/28

FACE N-Linked Oligosaccharide Profiling Kit

GK90000

TOOLSFOR GLYCOBIOLOGY

www.glyko.com


2/28


3/28

FACE N-Linked Oligosaccharide Profiling Kit (80 Reactions)

NOTE: We want successful results for our customers, so please read this entire booklet before starting

your experiment.Contents Page No

Kit Contents 2

Additional Reagents and Equipment Required 3

Introduction 4

Principle

Description of FACE N-linked Profiling System

Nature of FACE Oligo Standards and Controls

Summary of Protocol

Protocols

Section 1: Enzymatic Release of Oligosaccharides from Glycoproteins 9

Section 2: Labeling of Oligosaccharides from Glycoproteins 11

Section 3: Preparation of Samples and Standards for Electrophoresis 12

Section 4: FACE Electrophoresis 14

I. Preparation of OLIGO Gel Running Buffer

II. Set-up of FACEElectrophoresis Apparatus

III. Loading and Running FACE N-Linked Profiling Gels

Section 5: Processing of FACE N-Linked Profiling Gels 18

I. Gel Imaging using UV Transilluminator (lightbox)

II. Gel Imaging using FACE Imaging System

III. Gel Handling

IV. Oligosaccharide PreparationTroubleshooting Guide 21

Figures 23

Page 1


4/28


5/28

Additional Reagents and Equipment Required

FACE Electrophoresis Gel Box (ProZyme Product Code GK40026)

A CAP Type I water supply (such as Milli-Q)

Assorted pipeting devices including a 0-10 l capillary positive displacement pipette(e.g.Drummond Microdispenser, Fisher Cat# 21-176D)

Centrifugal vacuum evaporator

45C and 37C oven or water bath

Cold ethanol (100 %, nondenatured, -20C)

1.5 ml microcentrifuge tubes

Microfuge

UV-protective eye wear or face shield

UV transilluminator or FACE Imager

Page 3


6/28

INTRODUCTION

Complex carbohydrates are important components of all living things. In addition to providing energy

and structural supports for cells, increasing evidence has shown that the carbohydrate moieties of

glycoconjugates are often important as recognition determinants in receptor-ligand or cell-cell

interactions, in the modulation of immunogenicity and protein folding, and in the regulation of protein

bioactivity. Changes in the biological activity of glycoproteins often result from alterations in protein

glycosylation either through variable site occupancy or changes in the structure of the oligosaccharide

occupying a particular site. Using the FACE Imaging System, individual oligosaccharides can be

quantified to obtain molar ratios, amount of glycosylation and detect changes in the extent or nature of

glycosylation.

Principle

The FACE or "Fluorophore-Assisted-Carbohydrate-Electrophoresis" System is based on the use of

polyacrylamide gel electrophoresis to separate and quantify intact oligosaccharides released from

glycoproteins. FACE analysis begins with the release of Asparagine-linked (N-linked)

oligosaccharides from the glycoprotein using the enzyme N-Glycanase (Peptide N-glycosidase F). The

released oligosaccharides are then labeled with a fluorophore at the reducing termini by reductive

amination. The stoichiometry of labeling is such that only one molecule of fluorophore is attached to

each molecule of oligosaccharide. When labeling 20 nmoles or less of total sugar using the reagents and

labeling conditions described below, the fluorophore labeling efficiency is greater than 98%. Labelingmore than 20 nmoles of sugar in each reaction will result in reduced labeling efficiency. When labeling

more than 20 nmoles we recommend that you include the OLIGO Quantitation Control (E5) as an

internal labeling control. The fluorescent labeled oligosaccharides are then separated and quantified on

an OLIGO Profiling Gel using the FACEImaging System. Figure 1 (page 23) shows the profile of N-

linked oligosaccharides released from a number of glycoproteins.

Page 4


7/28

Description of the FACE N-linked OLIGO Profiling System

The FACE N-linked OLIGO Profiling Kit contains all reagents and buffers required to separate and

quantify the N-linked oligosaccharides released from glycoproteins and glycopeptides. This kit contains

all the components required to perform 80 enzymatic digests and 80 separate oligosaccharide labeling

reactions. In addition, the kit contains electrophoresis size markers for determining the relative

positions of oligosaccharide bands on the gel, and a control glycoprotein to verify the performance of

the enzyme and reagents in the kit. The kit also contains 10 precast OLIGO Profiling Gels and 5

OLIGO Gel Running Buffer packs sufficient to prepare 7.5 liters of Electrophoresis Running Buffer.

The FACE N-linked Oligo Profiling System involves four steps:

Step I) Release of the N-linked oligosaccharides from the glycoprotein, performed enzymatically.

Step II) Labeling of the mixture of released oligosaccharides with a fluorescent tag.

Step III) Separation of the fluorophore labeled oligosaccharides by polyacrylamide gel

electrophoresis. The resulting banding pattern represents the oligosaccharide profile of the glycoprotein.

This pattern can be thought of as a glycosylation "fingerprint" with the bands representing the

oligosaccharides released from the glycoprotein.

Step IV) Imaging of the gel. Imaging can be performed either on a UV lightbox to obtain qualitative

band conformation, or the gels can be imaged using the FACE Imaging System to determine the amount

of oligosaccharide present in each band and the relative mobility of the bands. Once separated on the

gel, individual oligosaccharide bands can be purified for further study.

Page 5


8/28

Nature of FACE OLIGO Standards and Controls

The OLIGO Ladder Standard (E3)

The OLIGO Ladder Standard (E3) consists of a mixture of glucose polymers ranging from Glucose 1to

greater than Glucose16. An illustration of the OLIGO Ladder Standard as it should appear at the

completion of electrophoresis is shown in Figure 4, Lane 1 (page 25). When the standard is prepared as

directed below and the recommended amount is loaded onto the gel, the band representing Glucose 4

contains 50 pmoles (25 pmoles for FACE imager users). The intensity of the Glucose4 band in the

standard mixture is less than the adjacent bands. This allows for easy identification of this band even if

the lower bands in the standard have run off the gel. Quantitation of the oligosaccharide bands in the

samples is achieved by comparing the intensity of the Glucose4 band with the intensity of sample bands

and it is therefore essential that the OLIGO Ladder Standard be present on each FACE OLIGO ProfilingGel. If the FACE Imaging System is used, dilute the E3 standard so that the Glucose4 band contains

25pmoles. Refer to the FACE Software Manual for a detailed description of band finding, quantitation

procedure, assigning a DP value, etc.

OLIGO Profiling Control (E4)

The OLIGO Profiling Control (E4) is trypsin inhibitor, used as a control for enzyme digestion and

fluorophore labeling. The oligosaccharide pattern for trypsin inhibitor is shown in Figure 4, Lane 2

(page 25). This control is included in the kit because:

If you use this control when you use the kit for the first time it will help you become familiar

with the kit procedures. If the profile you obtain looks like the pattern in Lane 2 of Figure 4 then

you can be assured that the kit is working properly.

If you have an unknown sample that may, or may not, contain N-linked oligosaccharides it is

often useful to include a tube of this control along with your tubes containing samples.

Following electrophoresis, if your samples do not show carbohydrate bands but you can see thenormal pattern of oligosaccharides released from the control, this will assure you that the

reagents are good and that the release and labeling procedures were performed properly.

Page 6


9/28

OLIGO Quantitation Control (E5)

The OLIGO Quantitation Control (E5) consists of 6 nmoles of unlabeled maltotetraose (Glucose4).

Once this control is reconstituted, 10 l of E5 will contain 200 pmoles of maltotetraose.

E5 may be used as an internal labeling control, provided that you verify that your sample does not

contain an oligosaccharide that will co-migrate with maltotetraose (e.g. Man3GlcNAc2 migrates very

close to Glucose4). As shown in Figure 3 (page 24), the labeling efficiency of your sample is greater

than 95% when labeling less than 20 nmoles. With some unknown samples you may not know how

much sugar you are labeling before you set up the digest. E5 is provided so that a "spike" of 200 pmoles

(10 l) may be added to the enzyme digest prior to the ethanol extraction step so that you can monitor

labeling efficiency. The maltotetraose band from the "spike" will be clearly visible on the gel (it will

co-migrate with the Glucose4 band in the OLIGO Ladder Standard (E3) as long as you load greater than1/40

thof the labeling reaction in the lane. After correcting for dilution of the labeling reactions you will

be able to determine the labeling efficiency of your sample by comparing the quantity of the internal

Glucose4 band against the same band in a lane containing (E5) labeled in a separate tube, or the

Glucose4 band in the lane containing the OLIGO Ladder Standard (E3). If you find that the quantity of

your spiked internal Glucose4 band (corrected for dilution) is only 190 pmoles (100% labeling efficiency

would give you 200 pmoles), the labeling efficiency of the sample is about 95%.

Page 7


10/28

Summary of Profiling Protocol

Purified Glycoprotein

N-Glycanase

Digestion 2 hours to overnightFree Oligosaccharides

Label with

fluorophore 3 hours to overnightLabeled Oligosaccharides

Load onto

FACEGel 30 minutesElectrophoresis 1 - 1 hours

FACEImaging and Data Analysis

Page 8


11/28

Protocols

SECTION 1

ENZYMATIC RELEASE OF OLIGOSACCHARIDES FROM GLYCOPROTEINS

NOTE: FACE Electrophoresis Buffer should be prepared up to one day in advance of the

electrophoresis run and stored at 4C. See page 14 for instructions.

1 Isolate the glycoprotein according to your usual procedures.

NOTE: The purified glycoprotein should be prepared in a phosphate buffer containing a minimum

amount of salt. Tris, acetate and citrate buffers will inhibit the labeling reaction.

2 If the volume of the glycoprotein solution required is greater than 100 l, dry the glycoprotein in a1.5 ml microcentrifuge tube. Generally 50-200 g of glycoprotein is required for analysis.

NOTE: The actual amount of glycoprotein required will depend on the size of the protein and the extent

of glycosylation. In general, at least 1 nmole of glycoprotein should be digested for analytical work, a

greater amount will be required for the preparation of purified oligosaccharides (see Page 19,

"NLinked Oligosaccharide Preparation from FACEProfiling Gels").

3 Add an equal volume of 2x Profiling Enzyme Buffer (C3) to the glycoprotein in solution or dissolve

the dried glycoprotein in 45 l of 1x OLIGO Profiling Enzyme Buffer made from the 2x (C3) stock

by adding an equal volume of water.

4 Remove a 45 l aliquot of the OLIGO Profiling Control (E4) and place in a 1.5-ml microfuge tube.

Store the remaining glycoprotein at 4C for future labeling controls.

5 Optional: SDS is often required to completely denature the glycoprotein prior to enzymatic

digestion. To denature add 2.5 l of SDS/-mercaptoethanol (C11), boil for 5 minutes and then add

2.5 l of NP-40 (C12) to both the samples and Oligo Profiling Control (E4) tube.

NOTE: Some proteins will precipitate when boiled, e.g. immunoglobulins. This procedure should be

used if your protein precipitates:

a. Add SDS/-ME at the recommended concentration. Incubate 5 minutes at room temperature.

b. Add NP-40 according to directions.

c. Add N-Glycanase and incubate overnight.

Page 9


12/28

6 Add 2 l of N-Glycanase (C1) to the glycoprotein sample and Oligo Profiling Control (E4). Mix

with finger flicks and centrifuge for 5 seconds. Store any remaining enzyme at 4C.

7 Incubate for 2 hours or overnight at 37C.

8 Prepare the OLIGO Quantitation Control (E5):

a. Add 300 l of distilled water to the tube labeled E5, which contains 6 nmoles of maltotetraose.

b. Resuspend the lyophilized carbohydrate by vortexing.

c. The concentration of the maltotetraose is now 200 pmoles/10 l.

9 Add 10 l (200 pmoles) of reconstituted E5 to the glycoprotein digest as an internal labeling control,

or label 10 l in a separate tube to check your quantitation according to the guidelines on page 4.

Store the unused reconstituted E5 at -20C.

10 Precipitate the protein by adding 3 volumes of cold ethanol. Keep samples on ice for 10minutes.

Spin samples in microcentrifuge for 5 minutes to pellet the protein.

11 Remove the supernatant and transfer to a clean 1.5 ml microcentrifuge tube.

DO NOT DISCARD THE SUPERNATANT; the released carbohydrates are present in the

supernatant.

12 If a large amount of protein was digested (>250 g), 5-10% of the released oligosaccharides may

remain in the pellet. The recovery of these oligosaccharides can be accomplished by drying the

pellet completely in a centrifugal vacuum evaporator or lyophilizer. Add 50 l water to resuspend,

then 150 l of cold ethanol and precipitate on ice. Centrifuge and combine the supernatants.

13 Dry supernatants in a centrifugal vacuum evaporator or lyophilize to a translucent pellet.

At this point samples may be stored at -20C, or proceed with the fluorophore labeling procedure

described in Section 2.

Page 10


13/28

SECTION 2

LABELING OF OLIGOSACCHARIDES FROM GLYCOPROTEINS

NOTE: Sample Handling and Storage

Always avoid exposing labeled samples and dyes to light or excessive heat.

Labeled samples are stable for 3 months when stored at -70C.

Unused solutions of the Labeling Dye and Labeling Reducing Agent may be stored for as long as

2 weeks at -70C. Thaw immediately before use.

I. Preparation of fluorophore labeling reagents

1 Add 125 l of the Oligo Labeling Diluent (L3) to one vial of Oligo Labeling Dye (L2). Mix well by

vortexing until dye is totally dissolved (warm at 37C if necessary). Centrifuge for 1 second in amicrocentrifuge. Reconstituted Labeling Dye solution may be stored for up to 2 weeks in the dark at

-70C.

2 Add 125 l of Labeling Solvent (L4) to one vial of Reducing Agent (L1). Mix well by vortexing

until crystals are completely dissolved, warm briefly if necessary. Reconstituted Reducing Agent

may be stored for 2 weeks at -70C.

II. Labeling reactions

Perform the following steps for the glycans released from your sample protein and the Oligo Profiling

Control (E4) (from Section 1 step 13). In addition, if you are using E5 as an external standard, take 10

l from the E5 tube, dry down in centrifugal vacuum evaporator or lyophilizer and follow these labeling

instructions.

1 Add 5 l of reconstituted Oligo Labeling Dye (L2) to each dried oligosaccharide pellet. Mix well

until the oligosaccharide pellet is dissolved.

2 Add 5 l of reconstituted Reducing Agent (L1). Mix well by vortexing. Centrifuge for 5 seconds

in a microfuge.

3 Incubate samples at 45C for 3 hours or overnight at 37C.

Page 11


14/28

SECTION 3

PREPARATION OF SAMPLE AND STANDARDS FOR ELECTROPHORESIS

I. Sample Preparation

1a Add 10 l water to your sample(s) and E4, then add 20 l of 2x Loading Buffer. Load 4 l or 1/10th

of the total sample volume directly onto the gel. Bring the E5 external control to a total volume of

16 l, with the addition of 6 l of 2x Loading Buffer (added to 10 l labeling reaction). Load 4 l or

50 pmoles onto the gel.

Alternatively,

1b Dry the samples, E4 and E5 in a centrifugal vacuum evaporator for approximately 15minutes oruntil the sample reaches a viscous gel stage.

2b Resuspend the fluorophore-labeled oligosaccharide from your sample only, in 10-20 l water.

NOTE: The actual volume of water used to resuspend the sample will depend on the amount of

oligosaccharide present in the sample. Start with 10 l, this will enable the sample to be diluted further

if necessary.

3b Remove 2 l of the sample and add 2 l of 2x Loading Buffer (E1).

4b Resuspend E4 in 20 l water and 20 l of 2x Loading Buffer. Resuspend E5 in 8 l water and 8 l

of 2x Loading Buffer.

5b Load 4 l of the sample, E4 and E5 directly onto the gel.

II. Preparation of OLIGO Ladder Standard (E3):

For non-imager users:

1a Resuspend the OLIGO Ladder Standard (E3) in 50 l of distilled water.

2a Add 50 l of 2x Loading Buffer (E1). After reconstitution the OLIGO Ladder Standard should be

stored at -70C.

Page 12


15/28

3a Load 4 l directly onto the gel; the maltotetraose band in the ladder should contain 50 pmoles.

For Imager users:

1b Resuspend the OLIGO Ladder Standard (E3) in 100 l of distilled water.

2b Add 100 l of 2x Loading Buffer (E1). After reconstitution, the OLIGO Ladder Standard should be

stored at -70C.

2c Load 4 l directly onto the gel; the maltotetraose band in the ladder should contain 25 pmoles.

Page 13


16/28

SECTION 4

FACE ELECTROPHORESIS

I. Preparation of 1.5 liter of FACEOligo Gel Running Buffer

(1.5 liter of buffer is sufficient for one electrophoresis run of one or two gels.)

NOTE: FACE electrophoresis buffer should be prepared up to one day in advance of the

electrophoresis run and stored at 4C.

1 Remove one of the Oligo Profiling Gel Running Buffer packs from the kit.

2 Cut open one end of the packet and carefully pour the contents into a 2-liter graduated cylinder and

add 1000 ml of distilled water.

3 Rinse buffer packet with 100 ml of water and add to the redissolved buffer.

4 Bring up to a final volume of 1.5 liter with water.

5 Mix well. The buffer must be chilled to 4-6C before use.

II. Set-up of FACE Electrophoresis Apparatus

FACE Electrophoresis of N-linked gels is best performed at a temperature range of 5-8C. We

recommend the use of a recirculating chiller, however satisfactory results can be obtained by chilling the

buffer prior to the electrophoresis run.

CAUTION: To prevent damage to the gel box, connect both quick disconnect fittings to gel box

before turning on the recirculator!

1 Place the FACE electrophoresis tank containing a stir bar on a mechanical stirrer. If using arecirculating chiller, connect it to the gel box cooling chamber with tubing and quick-connect

fittings. Turn on the circulator and stirrer; set the coolant temperature to 5C. Bleed all air out of

system by tilting the gel box in the direction of the outlet.

Page 14


17/28

2 Pour pre-cooled OLIGO Gel Running Buffer into the electrophoresis tank up to the level marked on

the outside of the box. The temperature of the buffer can be monitored during the run using a

thermometer inserted through the hole in the lid. The temperature will probably increase a few

degrees during electrophoresis but should not exceed 10C

III. Loading and Running FACEOLIGO Profiling Gels

1 Determine the number of gels required for the samples prepared. Each Oligo Profiling Gel contains

8 lanes. One outside lane should be used for the Oligo Ladder Standard (E3) leaving the other 7

lanes for samples. Optional: Load 2 l of the E2 tracking dye to an outside lane to monitor

electrophoresis. This leaves 6 lanes for samples. The FACEelectrophoresis box will accept one or

two gels.

2 Cut open 1 or 2 packages containing FACEOligo Profiling Gels. Gently remove the comb(s) from

the gel(s). To avoid distorting the wells, gently wiggle each comb to free the teeth from the gel, then

lift up slowly until the comb is released.

3 It is essential that the wells of the gel are thoroughly rinsed with Oligo Gel Running Buffer from the

upper buffer reservoir prior to sample loading. This is best accomplished by using a syringe with a

blunt needle (a Pasteur pipette is not recommended because of the possibility of breakage in the

wells).

4 Place the gel cassette(s), one on each side of the center core unit of the gel box with the short glass

plate against the gasket. Be sure the cassette is centered and that the cassette is resting on the "feet"

at the bottom of the apparatus. If running only one gel, place the buffer dam on the other side.

5 Place one wedge down each side of the cassette. Then push wedges down to obtain a seal between

the inner short plate and the gasket. Repeat this procedure on the other side. Both sides should now

be sealed against the gaskets.

6 Fill the upper buffer reservoir formed between the cassettes with approximately 100 ml of chilled

Oligo Gel Running Buffer and check for leaks. Final buffer level should be just below the electrode

supports. If a leak occurs, first check that the apparatus was assembled properly, then try pushing

down the wedge with slightly greater pressure. If the leak persists check the "Troubleshooting"

section of this manual.

Page 15


18/28

7 With the core unit containing the gels placed securely on the bench, load samples into the wells by

underlaying the upper buffer. Use flat sequencing pipette tips (e.g. Sigma T-1656) to load by

delivering the sample to the bottom of each well. Optimal resolution will be achieved by using 4 l

of sample per lane.

NOTE: For the most reliable quantitation of oligosaccharide bands the use of a positive displacement

pipette is recommended.

8 Place the core unit containing the loaded gels into the electrophoresis tank and place the lid on

electrophoresis tank.

9 First connect the power cords to the electrophoresis tank then connect the power supply. Connectthe positive (red) lead to the electrode marked with a red dot on the electrophoresis box. Connect

the negative (black) lead to the electrode marked with a black dot.

10 A thermometer may be placed into the lower buffer chamber through the hole in the lid to monitor

the temperature.

NOTE: The initial temperature of the lower buffer should be between 5C and 8C.

11 Turn on the power supply and select the proper current. FACEOLIGO Profiling Gels should be run

at a constant current of 20 mA per gel (40 mA for 2 gels). Limits on the power supply should be set

for 1000 volts and 60 watts. These run conditions will result in voltages of 100-400 V at the

beginning of the run and may approach 800 V at the end of the run. If the initial voltage is

significantly different, check to be sure that the leads are connected properly and that the buffers are

at the recommended levels.

12 Monitor electrophoresis by following the migration of the fast moving Orange Dye (E1). Generally,

electrophoresis is complete when the orange dye just exits the bottom of the gel in approximately1hour and 5 minutes. If the tracking dye (E2) is used, 4 colors should separate during the course of

the electrophoresis run.

13 Most N-linked oligosaccharides fall in the Glucose4 Glucose12 range in the OLIGO Ladder

Standard (E3) (see Figure 4, page 25) so the time of electrophoresis should be adjusted to optimize

the separation of this region of the gel.

Page 16


19/28

14 When the electrophoresis is complete, turn off the power supply. Disconnect the power cords from

the power supply and the electrophoresis tank. Turn off the recirculating chiller if used.

NOTE: If using the FACE Imager, begin warm-up now.

Page 17


20/28

SECTION 5

PROCESSING OF FACE OLIGO PROFILING GELS

Following Step 14, above, carefully remove the gels from the electrophoresis tank.

I. Gel Imaging using the UV Transilluminator (lightbox)

CAUTION: UV-protective eyewear or face shield should be worn. Avoid prolonged exposure to UV

light.

1 Allow UV lightbox to "warm-up" for at least 2 minutes in order to get maximum intensity output.

The lightbox must be long-wave UV (UVP Model TL-33 or equivalent) and have a peak output at

approximately 360 nm; this is not the type of box typically used for ethidium-stained DNA gels.

2 Optional: To increase sensitivity you may wish to peel the tape from the gel cassette, and

disassemble the cassette by carefully prying open the two glass plates, removing the gel completely

from the cassette and placing it directly on the UV lightbox.

Images of gels can be recorded using a Polaroid camera. The proper choice of light source, filters and

film must be made. A filter must be fitted to the camera lens, which completely covers the glass of the

lens, stray UV contacting the lens will cause it to fluoresce and subsequently lower the sensitivity of the

film. A Kodak Wratten Number 8 filter is suggested and can be purchased from Kodak and Kodak

distributors. A suitable filter will have no inherent fluorescence, peak transmission at approximately

500 nm, and bandwidth of 80 nm FWHM.

A medium speed, medium resolution, Polaroid film is recommended. Use Polaroid 53 film for cameras

that use single 4"x 5" sheet film; use Polaroid 553 film for cameras that use 8 sheet film cartridges.

Photographing Gels:

1 Photograph the gel using the lowest practical F setting on the lens, with the gel filling as much of

the frame as possible. In our experience, exposures at F5.6 using Polaroid 53 film have ranged

from 5 to 40 seconds using the equipment specified above. KEEP UV EXPOSURE OF THE

GEL TO A MINIMUM TO PREVENT BLEACHING.

Page 18


21/28

2 Develop film according to manufacturer's instructions.

II. Gel Imaging Using the FACE Imaging System

1 Turn on the FACE Imager and allow a warm-up period of at least 10 minutes before acquiring an

image. Allow the computer to initialize the Imager.

2 Clean the glass plates completely with lint-free tissue. A little distilled water should be used to

clean any obvious residue on the plates before imaging. The gel cassettes should be relatively dry

and free of dust before imaging.

3 Open the door to the Imager and place the cassette containing the gel into the cassette holder (see

the "FACE Software Manual" for imaging instructions).

III. Gel Handling

After imaging, gels may be processed in a number of ways depending on the needs of the investigator.

If the gel is no longer needed it should be properly discarded.

As long as the gel cassette is intact, it can be placed back in the electrophoresis apparatus and the

run continued in order to improve the resolution of the oligosaccharide bands.

Following imaging of the oligosaccharide gels, the glass plates can be separated and the gels

dried on a flat bed gel drier between sheets of Teflon membrane at 80C for 1 hour. After the gel

is dry, carefully peel the Teflon sheets away from the gel. Gels dried in this manner may be

stored indefinitely and re-imaged at any time.

NOTE: Drying fixes the bands so that they do not continue to diffuse through the gel.

IV. N-Linked Oligosaccharide Preparation from FACE Profiling Gels

FACEProfiling Gels may be used for the preparation of oligosaccharides (e.g. for subsequent enzymatic

sequencing) by loading aliquots of the same sample in all lanes. The following procedure is

recommended for maximum recovery of oligosaccharides. Due to loss of signal that may occur during

this procedure, accurate quantitation may not be possible on purified oligosaccharides.

Page 19


22/28

1 Following electrophoresis, bands on the gel can be visualized by placing the gel on a long wave UV

lightbox.

CAUTION: UV protective eyewear or face shield should be worn. Avoid prolonged exposure to UV

light.

2 Remove the tape and carefully separate the cassette with a broad spatula. Using a scalpel or razor

blade, carefully excise the band(s) of interest. This should be done as quickly as possible to

minimize photobleaching.

3 Place the gel slices in microcentrifuge tubes.

4 Soak the gel slices in a minimum volume of 100% ethanol for hour.

5 Pipet off the ethanol and suspend the gel slices in a minimum volume of water, enough to cover the

gel slices.

6 Vortex the gel slices gently, place at 4C overnight or all day (12-16 hours).

7 Remove the supernatant and place in a microcentrifuge tube.

8 If maximum recovery is required wash the gel slices, again, in a minimum volume of water. Place

the gel slices at 4C for 1 to 4 additional hours.

9 Remove the supernatant and pool with the first supernatant.

10 If any particulate material is visible, filter the supernatant pool through a 0.65 filter.

11 Dry the samples (supernatants).

Page 20


23/28

TROUBLESHOOTING GUIDE

Sample not moving or moving slowly on gel.

Leads may be reversed. Check leads to power supply and gel box. Check upper buffer level is above

the top of the short glass plate. Check the lower buffer level. If the level of the buffer is above the

orange gasket, remove 20-50 mls of buffer until the level is below the gasket.

Erratic voltage, voltage and/or current leak. (At the beginning of the run voltage is greater than

400V or readings are unstable.)

Make sure that the electrical leads are not arcing due to condensation on the lid of the tank. Dry the area

around the electrical posts on the inner box. Check that the leads to the power supply are connectedsecurely. Verify that the power supply is operating properly.

Band distortion in gel

From time to time band distortions may occur when running O-linked gels, for various reasons,

including:

Sample may be overloaded, use a maximum of 1/5th

of the volume of the labeling reaction for

each lane.

Wells may have been torn when comb was removed. Remove comb slowly using a gentle back

and forth rocking motion and lift vertically.

Smile effect on the gel may be the result of a gel not being cooled uniformly. Check that the

cooling system is on and working properly, and is free of air bubbles in the electrophoresis box.

Make sure that a stirring bar has been placed in the electrophoresis box and that the lower buffer

is being mixed. Check that power supply is set for proper current level - see Section 4-III-11 for

proper settings.

Fuzzy bands may be due to too high current used. Check Section 4-III-11 for proper settings.

Page 21


24/28

Upper buffer chamber leaks when cassettes are in place.

Check that the plates are clean, not cracked or chipped, and that they are centered on the inner core

assembly. Once the wedge has been brought into position against the glass, gently push down to engage

the sealing gasket. Check that the gaskets are not cracked and that they are seated properly in place (do

not grease gaskets). Pushing down with slightly greater pressure can stop most minor leaks.

If you are using a FACE Imaging System and would like us to take a look at your gel for

troubleshooting purposes, please e-mail a copy of the .raw or .IM2 file to us at [email protected]

Trademarks

FACE is a registered trademark of ProZyme, Inc., San Leandro, CA, USA

The following designations are trademarks owned by other companies:

Eppendorf is a trademark of Eppendorf Geratebau + Hinz, GmbH

Kodak is a trademark of the Eastman Kodak Company

Milli-Q is a trademark of the Millipore Corporation

N-Glycanase is a registered trademark of Genzyme Corporation, Boston, MA, USA. Recombinant

PNGase F is covered by US Patent No 5,238,821 and its foreign equivalents. The product is

manufactured under exclusive license by ProZyme, Inc

Nonidet is a trademark of Shell International Petroleum Company

Polaroid is a trademark of the Polaroid Corporation

Teflon is a trademark of E.I. DuPont de Nemours and Company

Tris is a trademark of Rohm and Haas

Page 22


25/28

Separation of N-Linked Oligosaccharides Released from Various Glycoproteins

Figure 1. Lane 1 FACE Oligo Ladder Standard

Lane 2 Fetuin N-linked oligosaccharides

Lane 3 HIV gp120 N-linked oligosaccharides

Lane 4 Human 1-acid glycoprotein

Lane 5 Bovine ribonuclease B

Lane 6 Human chorionic gonadotrophin

Lane 7 Human 1-trypsin

Lane 8 Bovine deoxyribonuclease

Page 23


26/28

Fluorophore Labeling

Figure 2. Fluorophore labeling by reductive amination

Figure 3. Fluorophore labeling efficiency of a di-sialylated biantennary oligosaccharide.

Page 24


27/28

1 2

Figure 4. Lane 1 E3FACE Oligo Ladder Standard

Lane 2 E4 Oligo Profiling Control

NOTE: The band marked in Lane 1 is Glucose4 , maltotetraose, which is provided individually as an

internal standard (E5).

Page 25


28/28

ProZyme, Inc.1933 Davis Street, Ste 207, San Leandro, CA 94577-1258 USA

Tel: 1(510) 638-6900 Toll-free: 1(800) 457-9444Fax: 1(510) 638-6919

E.mail: [email protected]

www.prozyme.com/glyko031804AC

n-linked oligosaccharide profiling manual

Documents