FACE N-Linked Oligosaccharide Profiling Kit GK90000

Transcription

1 FACE N-Linked Oligosaccharide Profiling Kit GK90000

2 TOOLS FOR GLYCOBIOLOGY Page 2

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4 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 FACE Electrophoresis 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 Preparation Troubleshooting Guide 21 Figures 23 Page 1

5 Kit Contents Catalog # Code Component Quantity Storage GK each Oligo Profiling Gels precast gels Room Temp WS0007 Oligo Gel Running Buffer 5 packs Room Temp on arrival GK packs GK packs 4 C (when reconstituted) E1 2x Loading Buffer 1 tube Room Temp E2 Tracking Dye 1 tube Room Temp C1 N-Glycanase 1 tube (100 mu) 4 C C3 2x Profiling Enzyme Buffer 1 tube 4 C C11 2% SDS/1M β-mercaptoethanol 1 tube 4 C C12 15% NP-40 1 tube 4 C L1 Reducing Agent (NaBH 3 CN) 2 tubes Room Temp on arrival -70 C (when reconstituted) L2 Oligo Labeling Dye (ANTS) 2 tubes Room Temp on arrival -70 C (when reconstituted) L3 Oligo Labeling Diluent 2 tubes Room Temp L4 Labeling Solvent (DMSO) 2 tubes Room Temp GK50006 E3 Oligo Ladder Standard 1 tube -20 C E4 Oligo Profiling Control 1 tube -20 C E5 Oligo Quantitation Control 1 tube -20 C on arrival -70 C (when reconstituted) Page 2

6 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 µl capillary positive displacement pipette (e.g. Drummond Microdispenser, Fisher Cat# D) Centrifugal vacuum evaporator 45 C and 37 C oven or water bath Cold ethanol (100 %, nondenatured, -20 C) 1.5 ml microcentrifuge tubes Microfuge UV-protective eye wear or face shield UV transilluminator or FACE Imager Page 3

7 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%. Labeling more 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 FACE Imaging System. Figure 1 (page 23) shows the profile of N-linked oligosaccharides released from a number of glycoproteins. Page 4

8 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

9 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 1 to greater than Glucose 16. 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 Glucose 4 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 Glucose 4 band with the intensity of sample bands and it is therefore essential that the OLIGO Ladder Standard be present on each FACE OLIGO Profiling Gel. If the FACE Imaging System is used, dilute the E3 standard so that the Glucose 4 band contains 25 pmoles. 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 the normal 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

10 OLIGO Quantitation Control (E5) The OLIGO Quantitation Control (E5) consists of 6 nmoles of unlabeled maltotetraose (Glucose 4 ). Once this control is reconstituted, 10 µl 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. Man 3 GlcNAc 2 migrates very close to Glucose 4 ). 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 µl) 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 comigrate with the Glucose 4 band in the OLIGO Ladder Standard (E3) as long as you load greater than 1/40 th of 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 Glucose 4 band against the same band in a lane containing (E5) labeled in a separate tube, or the Glucose 4 band in the lane containing the OLIGO Ladder Standard (E3). If you find that the quantity of your spiked internal Glucose 4 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

11 Summary of Profiling Protocol Purified Glycoprotein N-Glycanase Digestion 2 hours to overnight Free Oligosaccharides Label with fluorophore 3 hours to overnight Labeled Oligosaccharides Load onto FACE Gel 30 minutes Electrophoresis 1-1 ½ hours FACE Imaging and Data Analysis Page 8

12 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 4 C. 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 µl, dry the glycoprotein in a 1.5 ml microcentrifuge tube. Generally µg 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, "N Linked Oligosaccharide Preparation from FACE Profiling Gels"). 3 Add an equal volume of 2x Profiling Enzyme Buffer (C3) to the glycoprotein in solution or dissolve the dried glycoprotein in 45 µl of 1x OLIGO Profiling Enzyme Buffer made from the 2x (C3) stock by adding an equal volume of water. 4 Remove a 45 µl aliquot of the OLIGO Profiling Control (E4) and place in a 1.5-ml microfuge tube. Store the remaining glycoprotein at 4 C for future labeling controls. 5 Optional: SDS is often required to completely denature the glycoprotein prior to enzymatic digestion. To denature add 2.5 µl of SDS/β-mercaptoethanol (C11), boil for 5 minutes and then add 2.5 µl 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/β-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

13 6 Add 2 µl 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 4ºC. 7 Incubate for 2 hours or overnight at 37 C. 8 Prepare the OLIGO Quantitation Control (E5): a. Add 300 µl 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 µl. 9 Add 10 µl (200 pmoles) of reconstituted E5 to the glycoprotein digest as an internal labeling control, or label 10 µl in a separate tube to check your quantitation according to the guidelines on page 4. Store the unused reconstituted E5 at -20 C. 10 Precipitate the protein by adding 3 volumes of cold ethanol. Keep samples on ice for 10 minutes. 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 µg), 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 µl water to resuspend, then 150 µl 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 -20 C, or proceed with the fluorophore labeling procedure described in Section 2. Page 10

14 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 -70 C. Unused solutions of the Labeling Dye and Labeling Reducing Agent may be stored for as long as 2 weeks at -70 C. Thaw immediately before use. I. Preparation of fluorophore labeling reagents 1 Add 125 µl 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 37 C if necessary). Centrifuge for 1 second in a microcentrifuge. Reconstituted Labeling Dye solution may be stored for up to 2 weeks in the dark at -70 C. 2 Add 125 µl 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 -70ºC. 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 µl from the E5 tube, dry down in centrifugal vacuum evaporator or lyophilizer and follow these labeling instructions. 1 Add 5 µl of reconstituted Oligo Labeling Dye (L2) to each dried oligosaccharide pellet. Mix well until the oligosaccharide pellet is dissolved. 2 Add 5 µl of reconstituted Reducing Agent (L1). Mix well by vortexing. Centrifuge for 5 seconds in a microfuge. 3 Incubate samples at 45ºC for 3 hours or overnight at 37 C. Page 11

15 SECTION 3 PREPARATION OF SAMPLE AND STANDARDS FOR ELECTROPHORESIS I. Sample Preparation 1a Add 10 µl water to your sample(s) and E4, then add 20 µl of 2x Loading Buffer. Load 4 µl or 1/10 th of the total sample volume directly onto the gel. Bring the E5 external control to a total volume of 16 µl, with the addition of 6 µl of 2x Loading Buffer (added to 10 µl labeling reaction). Load 4 µl or 50 pmoles onto the gel. Alternatively, 1b Dry the samples, E4 and E5 in a centrifugal vacuum evaporator for approximately 15 minutes or until the sample reaches a viscous gel stage. 2b Resuspend the fluorophore-labeled oligosaccharide from your sample only, in µl 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 µl, this will enable the sample to be diluted further if necessary. 3b Remove 2 µl of the sample and add 2 µl of 2x Loading Buffer (E1). 4b Resuspend E4 in 20 µl water and 20 µl of 2x Loading Buffer. Resuspend E5 in 8 µl water and 8 µl of 2x Loading Buffer. 5b Load 4 µl 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 µl of distilled water. 2a Add 50 µl of 2x Loading Buffer (E1). After reconstitution the OLIGO Ladder Standard should be stored at -70 C. Page 12

16 3a Load 4 µl 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 µl of distilled water. 2b Add 100 µl of 2x Loading Buffer (E1). After reconstitution, the OLIGO Ladder Standard should be stored at -70 C. 2c Load 4 µl directly onto the gel; the maltotetraose band in the ladder should contain 25 pmoles. Page 13

17 SECTION 4 FACE ELECTROPHORESIS I. Preparation of 1.5 liter of FACE Oligo 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 4 C. 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 4-6 C before use. II. Set-up of FACE Electrophoresis Apparatus FACE Electrophoresis of N-linked gels is best performed at a temperature range of 5-8 C. 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 a recirculating 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 5ºC. Bleed all air out of system by tilting the gel box in the direction of the outlet. Page 14

18 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 10 C III. Loading and Running FACE OLIGO 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 µl of the E2 tracking dye to an outside lane to monitor electrophoresis. This leaves 6 lanes for samples. The FACE electrophoresis box will accept one or two gels. 2 Cut open 1 or 2 packages containing FACE Oligo 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

19 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. 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. 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 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 5 C and 8 C. 11 Turn on the power supply and select the proper current. FACE OLIGO 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 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 approximately 1 hour 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 Glucose 4 Glucose 12 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

20 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

21 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

22 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 80 C 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 FACE Profiling 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

23 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 ½ 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 4 C 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 4 C 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 0.65µ filter. 11 Dry the samples (supernatants). Page 20

24 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 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 connected securely. 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 1/5 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 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

25 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 a copy of the.raw or.im2 file to us at glyko@prozyme.com 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

26 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 α 1 -acid glycoprotein Lane 5 Bovine ribonuclease B Lane 6 Human chorionic gonadotrophin Lane 7 Human α 1 -trypsin Lane 8 Bovine deoxyribonuclease Page 23

27 Fluorophore Labeling Figure 2. Fluorophore labeling by reductive amination Figure 3. Fluorophore labeling efficiency of a di-sialylated biantennary oligosaccharide. Page 24

28 1 2 Figure 4. Lane 1 E3 FACE Oligo Ladder Standard Lane 2 E4 Oligo Profiling Control NOTE: The band marked in Lane 1 is Glucose 4, maltotetraose, which is provided individually as an internal standard (E5). Page 25

29 ProZyme, Inc Bay Center Place, Hayward, CA USA Tel: 1(510) Toll-free: 1(800) Fax: 1(510) E.mail: AD