FACE Monosaccharide Composition Kit GK TOOLS FOR GLYCOBIOLOGY

FACE Monosaccharide Composition Kit GK90100 TOOLS FOR GLYCOBIOLOGY www.glyko.com

FACE Monosaccharide Composition Kit (80 Reactions) Please read the following protocol right through before starting your experiment Contents Page No Kit Contents 2 Additional Reagents and Equipment Required 2 Introduction 3 Summary of Protocols 5 Protocols Section 1: Hydrolysis of Intact Glycoproteins 6 Section 2: Hydrolysis of Intact Oligosaccharides 9 Section 3: Fluorophore Labeling of Monosaccharides 12 Section 4: Preparation of Samples and Standards for Electrophoresis 14 Section 5: FACE Electrophoresis I. Preparation of MONO Gel Running Buffer 16 II. Set-up of FACE Electrophoresis Apparatus 16 III. Loading and Running FACE Mono Composition Gels 17 Section 6: Processing of FACE Mono Composition Gels I. Gel Imaging using U.V. Transilluminator (lightbox) 20 II. Gel Imaging using FACE Imaging System 20 III. Gel Handling 21 Section 7: Interpretation of Gel Images 22 Troubleshooting Guide 26 Figures 28 Page 1

Kit Contents Catalog # Code Component Quantity Storage GK60100 10 each WS0039 GK50102 2 packs GK50104 2 packs GK50106 Mono Composition Gels precast gels Room Temp Monosaccharide Gel Running Buffer 5 packs Room Temp on arrival 4 C when reconstituted G1 Mono 2X Loading Buffer 1 tube Room Temp E2 Tracking Dye 1 tube Room Temp M1 Hydrolysis Solution 1 1 tube Room Temp M2 Hydrolysis Solution 2 1 tube Room Temp M3 Re-N-Acetylation Reagent 1 tube Room Temp M6 Re-N-Acetylation Buffer 2 1 tube Room Temp L1 Labeling Reducing Agent 2 tubes Room Temp on arrival -70 C (when reconstituted) B2 Mono Labeling Dye, AMAC 2 tubes Room Temp on arrival -70 C (when reconstituted) B3 Mono Labeling Diluent 2 tubes Room Temp L4 Labeling Solvent 2 tubes Room Temp G3 Mono Ladder Standard 1 tube -20 C on arrival -70 C (when reconstituted) G4 Mono Composition Control 1 tube -20 C or -70 C G7 Glycoprotein Control 1 tube -20 C or -70 C Avoid exposing the MONO Composition gels, Mono Ladder Standard, and any fluorophore-labeled sample to excessive light or heat. Additional Reagents and Equipment Required FACE Electrophoresis Gel Box (GK40026) A CAP Type I water supply (such as Milli-Q) 1.5 ml and 0.5 ml conical polypropylene microcentrifuge tubes Cap locks for 0.5 ml conical polypropylene microcentrifuge tubes Centrifugal vacuum evaporator Assorted pipeting devices including a 5 µl positive displacement pipette Microcentrifuge Heating blocks Page 2

Introduction Overview of Monosaccharide Composition Using FACE The FACE or "Fluorophore-Assisted-Carbohydrate-Electrophoresis" system is based on the use of polyacrylamide gel electrophoresis to identify and quantify the monosaccharide composition of oligosaccharides. This information can be used to make predictions as to the nature of the oligosaccharide and the extent of oligosaccharide processing, as well as evaluating changes in monosaccharide composition. The FACE Monosaccharide Composition Kit contains the reagents and buffers required to hydrolyze the oligosaccharides of intact glycoproteins, isolated oligosaccharides, and synthetic oligosaccharides into component monosaccharides. All the components required to perform 80 separate hydrolysis and labeling reactions are provided. The kit contains a monosaccharide standard mixture of 6 monosaccharides that is used to identify and quantitate the concentration of neutral and amine monosaccharides present in the sample. The control glycoprotein and the control oligosaccharide can be used to verify the performance of the reagents in the kit. 10 precast MONO Composition Gels, and 10 MONO Gel Running Buffer packs sufficient to prepare 15 liters of electrophoresis running buffer are included. When performing these experiments it is important to remember that there is always some variability in the optimal conditions for acid hydrolysis. Therefore, we suggest you take 2 or more time points, label the samples and compare the intensities of the monosaccharides. The data from the hydrolysis time points that gives the greatest recovery of the monosaccharides is generally the most reliable. Determining the monosaccharide composition of a glycoprotein or purified oligosaccharide using the FACE Monosaccharide Composition System involves four steps: Step I) Hydrolysis- Sections 1 and 2 The oligosaccharides are hydrolyzed into component monosaccharides. Due to differences in the acid stability of various monosaccharides, two different hydrolysis conditions are recommended. The kit allows for the analysis of neutral and amine monosaccharides from isolated oligosaccharides or intact glycoproteins. Step II) Labeling- Section 3 The mixture of released monosaccharides is labeled with a fluorescent tag. See Figures 2 and 3, page 28 for a diagram of the labeling reaction and kinetics. Page 3

Step III) Separation- Sections 4 and 5 Separation of the fluorophore labeled monosaccharides is done by polyacrylamide gel electrophoresis. The resulting banding patterns represent the monosaccharide composition of the starting material. Step IV) Imaging- Sections 6 and 7 A qualitative determination of the monosaccharides present on the sample can be seen using a U.V. light box. A quantitative determination of the monosaccharides present can be obtained using the FACE Imaging System. Nature of FACE MONO Controls The Glycoprotein Control (G7) and the MONO Composition Control (G4) are included in the kit so the researcher can confirm the provided reagents and protocols are giving expected results. The use of these controls is optional but it is recommended that they be included along with unknowns until one is completely familiar with the FACE Monosaccharide Composition Analysis Kit. Note: To complete one Glycoprotein Control experiment and fully analyze the results it will require one MONO Composition Gel. Page 4

Summary of Monosaccharide Composition Protocol Glycoprotein or Purified Oligosaccharide Neutral Sugar Hydrolysis Amine Sugar Hydrolysis 5 Hours 1 or 3 hours Free Monosaccharides Label with fluorophore Overnight Labeled Monosaccharides Load onto FACE Gel 30 minutes Electrophoresis 2 hours FACE Imaging and Data Analysis Page 5

Protocols SECTION 1 HYDROLYSIS OF INTACT GLYCOPROTEINS Monosaccharides degrade at different rates when subjected to acidic condition. Different hydrolysis reactions are used to optimize the amount of a particular type of monosaccharide present. Two separate hydrolysis reactions are recommended for intact glycoproteins using 2N TFA for neutral sugars, and 4N HCl for amine sugars. If sufficient starting material is available, it is also recommended that a time course be performed for each hydrolysis. The hydrolysis conditions described below are appropriate for the provided controls and many samples. The protocols may need to be modified for optimal recovery and quantitation of monosaccharides from specific glycoprotein samples. Consistent hydrolysis is best achieved using a heat block with deep wells. The reaction tubes are placed inside the block, equilibrated to the specified temperature. An oven mitt placed over the heat block and weighted down makes a convenient oven and heats the reaction tube uniformly. Alternatively, a heat block filled with sand can be used. The reaction tubes should be buried deep in the sand to help prevent condensation. Condensation of the reaction in the top of the reaction tube can cause variable hydrolysis results. Glycoproteins should be dialyzed against distilled water because the amount of salt and type of buffer can interfere with hydrolysis. I. Preparation of Samples for Hydrolysis 1 Dialyze the glycoprotein sample against distilled water to remove buffers. Buffers interfere with hydrolysis by changing the final ph. 2 Use between 10 and 20 µg of dialyzed glycoprotein for each hydrolysis reaction (amine and neutral) in 50 µl of distilled water. Use 0.5 ml snap top tube with cap lock for hydrolysis. II. Preparation of Glycoprotein Control (G7) The Glycoprotein Control (G7) contains 120 µg of Bovine Fetuin, enough for 4 complete monosaccharide analyses. It should be used to become familiar with procedures and to monitor the efficiency of hydrolysis reactions, re-n-acetylation reactions, and fluorophore labeling reactions. It is recommended that 10 µg of the Glycoprotein Control be used for both of the hydrolysis reactions. Page 6

1 Resuspend Glycoprotein Control (G7) in 120 µl of distilled water. 2 Remove 10 µl for each of the two hydrolysis reactions. 3 Add 40 µl of distilled water to bring to a final volume of 50 µl. 4 Store the unused reconstituted (G7) at -20 C for future use. III. Determination of Neutral Sugar Content of Glycoproteins 1 Prepare 2X acid solution as follows: (enough for 4 separate glycoprotein analyses). CAUTION: M2 contains concentrated acid and should be handled with care Neutral Sugar Hydrolysis Solution Prepare 4N TFA by adding 90 µl TFA (M2) to 210 µl water 2 Add 50 µl of the 2X acid solution to one of the 50 µl glycoprotein samples prepared in Part I and one of the 50 µl glycoprotein control samples prepared in Part II. 3 Incubate the hydrolysis reaction as follows: Neutral Sugar Hydrolysis Conditions 100 C for 5 hours 4 After incubation, spin the reaction and cool for 30 minutes to 1 hour at -20 C. 5 Dry sample in a centrifugal vacuum evaporator (i.e. Speedvac) on "no heat" setting. Set aside until amine hydrolysis reaction is completed and dried, then proceed to Section 3 Fluorophore Labeling. IV. Determination of Amine Sugar Content of Glycoproteins 1 Prepare 2X acid solution as follows: (enough for 4 separate glycoprotein analyses) CAUTION: M1 contains concentrated acid and should be handled with care Page 7

Amine Sugar Hydrolysis Solution Prepare 8N HCl by adding 168 µl HCl (M1) to 83 µl water 2 Add 50 µl of the 2X acid solution to one of the 50 µl glycoprotein samples prepared in Part I and one of the 50 µl glycoprotein control samples prepared in Part II. 3 Incubate the hydrolysis reaction as follows: Amine Sugar Hydrolysis Condition 100 C for 3 hours 4 After incubation, spin the reaction and cool for 30 minutes to 1 hour at -20 C. 5 Dry sample in a centrifugal vacuum evaporator (i.e., Speedvac) on "no heat" setting. For amine hydrolysis reactions a re-n-acetylation step must be performed. 6 Resuspend the dried monosaccharide pellet in 10 µl of Re-N-acetylation Buffer 2 (M6). Vortex the sample. 7 Add 1 µl of Re-N-acetylation Reagent (M3). Vortex the sample. 8 Incubate on ice for 15 to 30 minutes. 9 Dry sample in a centrifugal vacuum evaporator on "no heat" setting. Set aside until neutral hydrolysis reaction is completed and dried, then proceed to Section 3 Fluorophore Labeling Page 8

SECTION 2 HYDROLYSIS OF ISOLATED OLIGOSACCHARIDES I. Preparation of Samples for Hydrolysis 1 Use approximately 300 pmoles of oligosaccharide per each hydrolysis reaction (amine, and neutral) in 50 µl of distilled water. Use a 0.5 ml snap top tube with cap lock for hydrolysis. II. Preparation of MONO Composition Control (G4) The MONO Composition Control (G4) contains 4 nmoles of N-acetyllactosamine and should be used to monitor the efficiency of the amine hydrolysis reactions, re-n-acetylation reactions, and fluorophore labeling reactions. It is recommended that 1 nmole of the MONO Composition Control (G4) be used as a reagent control each time a new vial of MONO Labeling Dye (B2) and Labeling Reducing Agent (L1) are reconstituted. 1 Add 200 µl of distilled water to the MONO Composition Control (G4) and vortex. Centrifuge for 1 second. 2 Transfer 50 µl of the reconstituted MONO Composition Control (G4) to a 0.5 ml snap top tube with cap lock for use in amine hydrolysis. 3 The remaining 150 µl of MONO Composition Control (G4) should be stored at -20 C for future use. III. Determination of Neutral Sugar Content of Glycoproteins 1 Prepare 2X acid solution as follows: (enough for 4 separate isolated oligosaccharides analyses) CAUTION: M2 contains concentrated acid and should be handled with care Neutral Sugar Hydrolysis Solution Prepare 4N TFA by adding 90 µl TFA (M2) to 210 µl water Page 9

2 Add 50 µl of the 2X acid solution to one of the 50 µl isolated oligosaccharide samples prepared in Part I. 3 Incubate the hydrolysis reaction as follows: Neutral Sugar Hydrolysis Conditions 100 C for 5 hours 4 After incubation, spin the reaction and cool for 30 minutes to 1 hour at -20 C. 5 Dry sample in a centrifugal vacuum evaporator (i.e., Speedvac) on "no heat" setting. Set aside until amine hydrolysis reaction is completed and dried, then proceed to Section 3 Fluorophore Labeling. IV.Determination of Amine Sugar Content of Isolated Oligosaccharides 1 Prepare 2X acid solution as follows: (enough for 4 separate isolated oligosaccharide analyses) CAUTION: M1 contains concentrated acid and should be handled with care Amine Sugar Hydrolysis Solution Prepare 8N HCl by adding 168 µl HCl (M1) to 83 µl water 2 Add 50 µl of the 2X acid solution to one of the 50 µl isolated oligosaccharides samples prepared in Part 1 and the MONO Composition Control (G4) control prepared in Part 2. 3 Incubate the hydrolysis reaction as follows: Amine Sugar Hydrolysis Condition 100 C for 1 hours 4 After incubation, spin the reaction and cool for 30 minutes to 1 hour at -20 C. 5 Dry sample in a centrifugal vacuum evaporator (i.e., Speedvac) on "no heat" setting. Page 10

For amine hydrolysis reactions a re-n-acetylation step must be performed. 6 Resuspend the dried monosaccharide pellet in 10 µl of Re-N-acetylation Buffer 2 (M6). Vortex the sample. 7 Add 1 µl of Re-N-acetylation Reagent (M3). Vortex the sample. 8 Incubate on ice for 15 to 30 minutes. 9 Dry sample in a centrifugal vacuum evaporator on "no heat" setting. Set aside until neutral hydrolysis reaction is completed, then proceed to Section 3 Fluorophore Labeling. Page 11

SECTION 3 FLUOROPHORE LABELING OF MONOSACCHARIDES Note: Sample Handling and Storage Always avoid exposing labeled samples and dyes to light or excess heat. Labeled samples are stable when stored for 3 months at -70 C. Unused solutions of the dye and reducing agent can be stored for as long as 2 weeks at -70 C. Thaw immediately before use. I. Preparation of Fluorophore Labeling Reagents 1 Add 75 µl of Labeling Solvent (L4) to one vial of MONO Labeling Dye (B2). Mix well by vortexing until dye is totally dissolved. Centrifuge for 1 second in microcentrifuge. Reconstituted Labeling Dye (B2) is stable for 2 weeks when stored at -70 C. NOTE: Transfer the remaining unused Labeling Solvent (L4) to a clean Eppendorf (or equivalent) tube and save for use in Section 4. NOTE: THE DIRECTIONS FOR STEP 2 HAVE REVERTED TO ORIGINAL INSTRUCTIONS! In recent versions of this kit, vial L1 contained more Reducing Agent than before and the instructions were modified so that a reconstituted Labeling Reducing Agent was no different from that prepared according to previous versions of this kit. The Labeling Reducing Agent (L1) now contains the same amount as the original version of this kit, thus the kit instructions have now reverted to the original instructions. 2. Add 125 µl of Labeling Solvent (L4) to one vial of Labeling Reducing Agent (L1). Mix well by vortexing until crystals are completely dissolved, warming briefly at 37 C if necessary. Reconstituted Labeling Reducing Agent is stable for 2 weeks when stored at -70 C. NOTE: STEP 3 HAS BEEN REMOVED MONO Labeling Diluent is now supplied as one vial! In recent versions of this kit, MONO Labeling Diluent previously supplied as vial B3 was replaced by B3a (Water) and B3b (Acetic Acid). MONO Labeling Diluent is now supplied as a single vial (B3) and may be used directly (no mixing is required). Page 12

II. Labeling of Samples and Controls (G4 and G7) 1 Add 2.5 µl of the MONO Labeling Diluent (B3) to each dried monosaccharide pellet. Store unused MONO Labeling Diluent (B3) at room temperature. 2 Mix well until any visible pellet is dissolved. 3 Add 2.5 µl of reconstituted MONO Labeling Dye (B2). 4 Add 5 µl of reconstituted Labeling Reducing Agent (L1). Vortex to mix well. Centrifuge for one second in microcentrifuge. 5 Best results are obtained by incubating samples at 37 C overnight (approximately 16 hours) for quantitative labeling, although samples can be labeled at 45 C for 2 hours (See Figure 3, page 28). CAUTION: Temperatures in excess of 45 C during drying will cause degradation of the monosaccharides. 6 Dry samples in centrifugal vacuum evaporator using low or no heat for approximately 15 minutes or until the sample reaches a viscous stage. Do not dry the sample completely or it will be difficult to resuspend. 7 Proceed to Section 4 Preparation of Samples and Standards for Electrophoresis. Page 13

SECTION 4 PREPARATION OF SAMPLE AND STANDARDS FOR ELECTROPHORESIS Standards are prepared by Glyko to give reliable performance. However due to circumstances such as, improper storage, and numerous freeze-thaw cycles, the values of the standards may become inaccurate. To minimize loss of accuracy in quantitation follow the recommended storage conditions and limit freeze-thaw cycles. Electrophoresis should be performed on the samples immediately after resuspension. Storage of the resuspended samples can result in the appearance of an artifact band that migrates slightly slower than mannose in the standard ladder. Resuspend the dried sample immediately before electrophoresis is to be done. This will minimize the appearance of the band. Store dried labeled samples at -70 o C. I. Recommended Preparation of Intact Glycoproteins and Isolated Oligosaccharide Hydrolysis Reactions Due to large differences in the amounts of monosaccharides released from intact glycoproteins or isolated oligosaccharides, the following recommended volumes for sample preparations may need to be varied to optimize the loading of a particular sample. Optimal resolution will be achieved by loading 4 µl in a lane. 1 Resuspend the dried, labeled monosaccharides prepared in Section 3 in 5 µl Labeling Solvent (L4). 2 Add 15 µl of water and mix. 3 Remove a 3 µl aliquot to a second tube and add 3 µl of MONO 2X Loading Buffer (G1) to give 6 µl of a 1:1 dilution of sample in loading buffer. 4 Load 4 µl of a resuspended sample in a lane. This is equivalent to 1/10 th of the total monosaccharides released in that reaction tube. Page 14

II. Preparation of MONO Ladder Standard 2 (G3) The MONO Ladder Standard 2 (G3) consists of a mixture of fluorophore labeled monosaccharides: N-acetylgalactosamine, glucose, galactose, mannose, fucose and N-acetylglucosamine. The sample lanes are compared to the MONO Ladder Standard 2 lane to identify and quantify the amount of monosaccharides in the sample. It is essential that the MONO Ladder Standard 2 is present on each FACE MONO Composition Gel. The migration of each of the monosaccharides in the MONO Ladder Standard 2 is shown in Figure 2, Lane 1, on page 24. 1 Resuspend the MONO Ladder Standard 2 (G3) in 5 µl of Labeling Solvent (L4). 2 Add 45 µl of water. 3 Add 50 µl of MONO 2X Loading Buffer (G1) to give a total volume of 100 µl. 4 Load 4 µl in a lane to give 100 pmoles of each monosaccharide in the MONO Ladder Standard 2. See Figure 1, lanes 1 and 7, page 24. If only a small amount of monosaccharide is present in your sample then make a 1:1 dilution of resuspended (G3) in 1X Loading Buffer and use 4 µl, which results in 50 pmoles per band. III. Preparation of the MONO Composition Control (G4) and Glycoprotein Control (G7) Hydrolysis Reactions A gel showing the expected results can be found in Figure 1, page 24. 1 Resuspend the dried, labeled controls prepared in Section 3 in 5 µl Labeling Solvent (L4). 2 Add 15 µl of water and mix. 3 Remove a 3 µl aliquot to a second tube and add 3 µl of MONO 2X Loading Buffer (G1) to give 6 µl of a 1:1 dilution of sample in loading buffer. 4 Load 4 µl in a lane, which is equivalent to 1/10 th of the total monosaccharides released in that reaction tube. Page 15

SECTION 5 FACE ELECTROPHORESIS FACE Electrophoresis must be performed at a starting buffer temperature of 5-8 o C. I. Preparation of 1.5 liter of MONO Gel Running Buffer (1.5 liter of buffer is sufficient for one electrophoresis run of one or two gels.) 1 Remove one packet of MONO Gel Running Buffer 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. (Use cold water if electrophoresis is going to be performed immediately). 3 Rinse buffer packet with 100 ml of water and add this rinse to the 1000 ml. 4 Bring up to a final volume of 1.5 liter with water. 5 Mix well and refrigerate at 5-8 C. Note: The running buffer may be made in advance and stored at 4 C for two weeks. If a precipitate forms, the buffer should be discarded. II. Set-up of FACE Electrophoresis Apparatus CAUTION: To prevent damage to the gel box, connect both quick disconnect fittings to gel box before turning on recirculator! 1 Place the FACE electrophoresis tank containing a stir bar on a mechanical stirrer. Connect the gel box cooling chamber to a recirculating chiller with tubing and quick-connect fittings. Turn on the circulator and stirrer and set the coolant temperature to 5 C. Bleed all air out of system by tilting the gel box in the direction of the outlet. 2 When the coolant has equilibrated to temperature, pour cooled MONO Gel Running Buffer into the electrophoresis tank up to the fill line indicated on the side of the electrophoresis box. The Page 16

temperature of the buffer can be monitored during the run using a thermometer inserted through the hole in the lid III. Loading and Running FACE MONO Composition Gels 1 Determine the number of gels required for the samples prepared. Each MONO Composition Gel contains 8 wells. One outside lane should be used for the Ladder Standard (E3) leaving the other 7 lanes for samples. The outside wells should be used for MONO Ladder Standard (G3) and Tracking Dye (E2) leaving the 6 inner wells for samples. The electrophoresis core unit will accept one or two gels. 2 Cut open 1 or 2 packages containing MONO Composition Gel cassettes. If you are running only one gel, use the buffer dam on the other side. 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 out with MONO 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 pipet is not recommended because of the possibility of breakage into 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 you are 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 MONO Gel Running Buffer and check for leaks. Final buffer level should be just below the electrode supports. If a leak occurs first check to be sure the apparatus was assembled properly, then try pushing down the wedges with slightly greater pressure. If the leak persists check the troubleshooting section of this manual. Page 17

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 µl of sample per lane. Note: For the most reliable quantitation of oligosaccharide bands the use of a positive displacement pipette is recommended. Recommended loading volumes: MONO Ladder Standard (G3) A load of 4 µl of (G3) standard in a lane will result in 100 pmoles of monosaccharide in each band. If the sample being analyzed contains small amounts of carbohydrate, dilute the reconstituted MONO Ladder Standard 2 (G3) with 1X MONO Loading Buffer and load 4 µl into wells. Tracking Dye (E2) Load 2 µl in a lane from the vial. MONO Composition Control (G4) Load 4 µl of the reconstituted labeled control. This results in approximately 100 pmoles of GlcNAc. Galactose degrades under the harsh amine hydrolysis and will possibly be less than GlcNAc Glycoprotein Control (G7) Load 4 µl of each reconstituted hydrolysis reaction in separate wells. Results can be found in Figure 1, page 24. Samples. Load 4 µl of each labeled monosaccharide sample in a lane. If the gel appears to be overloaded use less sample but always adjust sample volume to 4 µl with 1X MONO Loading Buffer before loading into well. 8 Place the core unit containing the loaded gels into the electrophoresis tank and place the lid on. 9 First connect the power cords to the electrophoresis tank then connect the power supply. Connect the 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 can 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 5 C and 8 C. Page 18

11 Turn on the power supply and select the proper current. FACE MONO Profiling Gels should be run at a constant current of 30 ma per gel (60 ma for 2 gels, 30mA for 1 gel and buffer dam). Limits on the power supply should be set for the maximum volts and watts. Starting Conditions One Gel and One Buffer Dam Two Gels Temp = 5-8ºC Temp = 5-8ºC Volts = 400-600 Volts = 400-600 mamps = 30 mamps = 60 Watts = 15-25 Watts = 30-40 End of Run Conditions Temp = 15-20ºC Temp = 22-30ºC Volts = 800-1200 Volts = 850-1000 mamp = 30 mamp = 60 Watts = 25-40 Watts = 50-65 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 tracking dyes. At the end of the run the blue dye in the loading buffer should run near or off the bottom of the gel and the red dye in the tracking dye should have migrated to a point about ½ way down the gel. Run times generally are 1 to 1.5 hours. If using the FACE Imaging System turn on the Imager approximately 5 minutes prior to removing the gel 13 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. 14 Proceed to Section 6 to image immediately! Page 19

SECTION 6 PROCESSING OF FACE MONO COMPOSITION GELS Following Step 14, above, carefully remove the gels from the electrophoresis tank I. Gel Imaging using the U.V. Transilluminator (lightbox) CAUTION: U.V. protective eyewear or face shield should be worn. Avoid prolonged exposure to U.V. light. For direct visual observation of AMAC labeled carbohydrate band patterns on the U.V. light box (368nm Transilluminator) it is advisable to wear protective eye wear that blocks not only ultraviolet light but also visible blue light. Blocking the visible blue light dramatically improves the visibility of bands. Glyko suggests using very economical UV absorbing goggles such as Oberon Model 7020 (Oberon Co., New Bedford, MA) or their equivalent. 1 Depending on your light source you may be able to visualize the fluorescent bands through the cassettes on the light box (as with the UVP Chromato-Vue TL-33). If the background is high and you cannot see the bands, rinse out the wells of the gel with distilled water to remove excess fluorescent dye, then peel the tape off the gel cassettes, carefully pry open the plates, and lay the gel on the light box. 2 When viewing and photographing the gel on a UV light box the best results will be obtained using a long wavelength UV transilluminator with peak light output at 370nm and a Wratten 57A filter on the camera. It is our experience that any setup designed to photograph EtBr stained DNA gels (Orange filter, peak transmission at 515nm, FWHM = 80nm) should give acceptable results when viewing and photographing FACE gels. (Filters should be of the absorption type and not interference filters). 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. Page 20

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, the gels can 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 monosaccharide bands. Following imaging of the monosaccharide gels, the glass plates can be separated and the gels dried on a flat bed gel drier between sheets of Teflon membrane at 80 C for 1 hour. After the gel is dry, carefully peel the Teflon sheets away from the gel. Gels dried in this manner can be stored indefinitely and re-imaged at any time. Page 21

SECTION 7 INTERPRETATION OF GEL IMAGES The FACE MONO Composition system is used to separate, identify, and quantify monosaccharides released form glycoproteins and purified oligosaccharides. The first step in gel interpretation is to become familiar with the MONO Ladder Standard 2 (G3). The MONO Ladder Standard 2 contains a mixture of six common monosaccharides found in animal cells: N-acetylgalactosamine, mannose, fucose, glucose, galactose, and N-acetylglucosamine. The six monosaccharides have a characteristic and reproducible banding pattern on MONO Composition Gels (See Figure 1, lanes 1 and 7, page 24). Each of the six monosaccharide bands in the MONO Ladder Standard 2 contains 100 pmoles of each particular monosaccharide when reconstituted as specified in Section 4. The identification and quantitation of the monosaccharides released in the sample during hydrolysis is made by comparing the position and fluorescent intensity of the monosaccharides in the sample with the corresponding monosaccharides in the standard lane. Neutral monosaccharides may appear in the amine hydrolysis reaction. Due to the harsh acid conditions neutral monosaccharides in the amine hydrolysis are not to be used for quantitative purposes. The appearance of glucose in the sample does not necessarily mean the glycoprotein sample contains glucose. The presence of glucose in the sample can result from the hydrolysis of cellulose from wipes, powdered gloves, or cloth. Detergents like octyl-glucoside, some protein cross-linkers, or protein column material (Sepharose, etc.) are also a source of glucose contamination. Example of a FACE Monosaccharide Composition Experiment: In the following example a monosaccharide analysis will be performed on the Glycoprotein Control (G7). Purpose of Experiment: To determine type of monosaccharides present on a glycoprotein and whether the glycoprotein contains O-linked oligosaccharides. Procedures: Follow protocols to hydrolyze the monosaccharides attached to the protein(section 1). Label the released monosaccharides (Section 3) and run the labeled monosaccharides on a MONO Composition Gel (Sections 4 and 5). Visualize the separated monosaccharides in the gel (Section 6). Page 22

Results: The results can be interpreted using two different methods, a U.V. light box or the FACE Imaging system 1 2 3 4 5 6 7 Figure 1. Lane 1 Monosaccharide Standards Lane 2 Fetuin amine hydrolysis Lane 3 Glucose and GlcNAc Lane 4 Fetuin neutral hydrolysis Lane 5 Glucose and GlcNAc Lane 6 N-acetyllactosamine monosaccharides Lane 7 Monosaccharide Standards I. Using a UV Light box 1 Refer to Figure 1 above Lane 2 contains the amine hydrolysis results, find a band migrating high in the gel which migrates consistent with GalNAc in the standard lane. This confirms that the protein contains GalNAc and indicates probable O-linked oligosaccharides. GlcNAc, found in N-linked oligosaccharides also appears in the same lane. Determine the type of neutral monosaccharides on the glycoprotein by comparing the neutral hydrolysis results in lane 4, with the MONO Ladder Standard 2 (G3) in lane 7. The gel shows that the neutral sugars galactose and mannose are present. II. Using the FACE Imaging System 1 Refer to Figure 1 to interpret the banding patterns on the gel follow the description given above. In addition to visualizing the types of monosaccharides present on the glycoprotein, quantitation and Page 23

ratios of monosaccharides can be determined using the data in the band table. Perform a "band finding" routine and standard lane analysis according to instructions in the FACE Software Manual. To quantitate the monosaccharides from the amine and neutral hydrolysis use the MONO Ladder Standard 2 (lane 7). Quantitation of a Glycoprotein Control resulted in the following monosaccharide values (pmoles) GalNAc Lane 2 (49.91-24.05)=25.86* Mannose Lane 4 81.14 Fucose Lane 4 0.00 Glucose Lane 4 12.01 Galactose Lane 4 129.08 GlcNAc Lane 2 190.49 *Note: Dye contaminants sometimes migrate close to or with GalNAc. If this is the case the band finding routine may include these bands in the quantitation. The value from the contaminants in lane 4, was subtracted from the GalNAc value given in Lane 2 to find the accurate quantity of GalNAc. 1 To determine the ratio of monosaccharides on the glycoprotein the following procedure is suggested: a. Sum the pmoles given in the band table: GlcNAc and GalNAc values from amine hydrolysis reaction, neutral monosaccharide values from neutral hydrolysis (in this example glucose is considered a contaminant and its value is excluded). b. Divide each monosaccharide amount by the total amount of monosaccharide present. Using the above quantities, excluding the glucose value, the following ratios of monosaccharides were obtained for the Glycoprotein Control: GalNAc 6% Mannose 19% Galactose 30% GlcNAc 45% 3 To estimate the total weight of carbohydrate present on the glycoprotein sample and determine the percent glycosylation, follow the outline below: a. Multiply the pmoles values for the monosaccharides given in the band table by the dilution of the sample loaded onto the gel: GlcNAc and GalNAc values from amine hydrolysis reaction Page 24

(neutral sugars in the amine hydrolysis lane are ignored), neutral monosaccharide values from neutral hydrolysis.(in this example glucose is considered a contaminant and its value is excluded). The dilution factor for the Glycoprotein Control is 10. This gives the total pmoles of each monosaccharide present in the reaction tube. b. Multiply each monosaccharide's total pmoles/reaction tube e.g. [Molecular weight of galactose =180.2] - [H 2 O=18.0] = 162.2 When monosaccharides are hydrolyzed they gain a water molecule that is not present on the original oligosaccharide. This gives the total weight of each individual monosaccharide in the sample. c. Sum the total weight of each of the monosaccharides present in the sample to estimate the total weight of carbohydrate (less sialic acid). d. Divide this total weight of carbohydrate present on the glycoprotein by the total weight of glycoprotein used in a single hydrolysis reaction. In the Glycoprotein Control 10 µg is used in each hydrolysis reaction therefore total carbohydrate divided by 10 µg= percent carbohydrate. e. The FACE monosaccharide kit is not an effective method to analyze sialic acids. To determine sialic acid content we recommend the use of our DMB labeling kit (Cat No K-407) for more effective and reproducible labeling of sialic acids prior to separation on reverse phase HPLC. Once sialic acid content has been determined then insert the quantity into the calculations used in the FACE monosaccharide analysis Using the above procedure, excluding the glucose value, the following values were used to estimate percent glycosylation of the Glycoprotein Control: Monosaccharide Dilution pmoles carbohydrate/rxn. Mol. Weight µg carbohydrate/rxn. Factor (-H2O) GalNAc 10 259 203.2 0.053 Mannose 10 811 162.2 0.132 Galactose 10 1291 162.2 0.209 GlcNAc 10 1905 203.2 0.387 Total µg of carbohydrate= 0.781 Total µg of glycoprotein=10 The percent glycosylation minus sialic acid (the total µg of carbohydrate divided by the total µg of glycoprotein) =7.8% Page 25

TROUBLESHOOTING GUIDE "No load" light flashes on power supply. Verify that the power supply is operating properly and that buffers are at the recommended levels. Band distortion in gel From time to time band distortions can occur when running O-linked gels. There may be various reasons including: Sample may be overloaded, use a maximum of 1/5 th 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 gel can be a result of the 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 5-III-11 for proper settings. Fuzzy bands may be due to too high current used. Check Section 5-III-11 for proper settings. 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 connected securely. Verify that the power supply is operating properly. 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. Page 26

Upper buffer chamber leaks when cassettes are in place. Check that the plates are clean and 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. "Incomplete hydrolysis". Released monosaccharides are lower than expected amount. Buffer components present in sample. Sample must be desalted or dialyzed to remove all buffer before hydrolysis. Trademarks FACE is a registered trademark of ProZyme, Inc The following designations are trademarks owned by other companies. Chromato-Vue is a trademark of UVP, Inc 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 Nonident is a trademark of Shell International Petroleum Company Polaroid is a trademark of the Polaroid Corporation Sepharose is a trademark of Amersham Pharmacia Biotech Teflon is a trademark of E.I. DuPont de Nemours and Company Tris is a trademark of Rohm and Haas Page 27

Fluorophore Labeling Figure 2. Fluorophore Labeling of Monosaccharides by Reductive Amination Figure 3. Fluorophore Labeling of 5 nmoles of glucose. Page 28

ProZyme, Inc. 1933 Davis Street, Ste 207, San Leandro, CA 94577-1258 USA Tel: 1(510) 638-6900 Fax: 1(510) 638-6919 E.mail: glyko@prozyme.com www.prozyme.com/glyko 090304AB