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Separation and Purification of Glycosaminoglycans (GAGs) from Caenorhabditis elegans
秀丽隐杆线虫糖胺聚糖(GAG)的分离和纯化   

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Scientific Reports
Oct 2016

Abstract

The nematode Caenorhabditis elegans is a popular model organism for studies of developmental biology, neurology, ageing and other fields of basic research. Because many developmental processes are regulated by glycosaminoglyans (GAGs) on cell surfaces and in the extracellular matrix, methods to isolate and analyze C. elegans GAGs are needed. Such methods have previously been optimized for other species such as mice and zebrafish. After modifying existing purification protocols, we could recently show that the nematodes also produce chondroitin sulfate, in addition to heparan sulfate, thus challenging the view that only non-sulfated chondroitin was synthesized by C. elegans. We here present our protocol adapted for C. elegans. Since the purification strategy involves separation of non-sulfated and sulfated GAGs, it may also be useful for other applications where this approach could be advantageous.

Keywords: Glycosaminoglycans (糖胺聚糖), Caenorhabditis elegans (秀丽隐杆线虫), Proteoglycans (蛋白多糖), Ion exchange chromatography (离子交换色谱法), Sulfation (硫酸化)

Background

Glycosaminoglycans (GAGs) are linear polysaccharide chains of repeating disaccharide units, which are often substituted with sulfate groups. Except for hyaluronan, which is a non-sulfated GAG synthesized at the plasma membrane without being anchored to any protein, all other GAGs are covalently linked to core proteins, thus forming proteoglycans (PGs). The most common GAGs found on PGs are heparan sulfate (HS) and chondroitin sulfate (CS)/dermatan sulfate, containing N-acetyl-glucosamine and N-acetyl-galactosamine, respectively (Zhang, 2010). During their biosynthesis in the Golgi compartment, which is a non-template driven process, they are subject to multiple modifications, including epimerization of glucuronic acid into iduronic acid and sulfation at various positions (Bulow and Hobert, 2006; Zhang, 2010). The sulfation patterns generated are important for the ability of the GAG chains to interact with growth factors and cytokines, which in turn is essential for their ability to influence development and other important physiological processes (Bishop et al., 2007).

In order to analyze their composition, GAGs need to be purified from crude cell or tissue lysates, most commonly achieved by ion exchange chromatography after protease and nuclease digestion (Ledin et al., 2004). Disaccharides, generated by the action of specific GAG lyases, can then be identified using different chromatography methods or mass spectrometry (Shao et al., 2013; Kiselova et al., 2014). We and several other labs have used reversed-phase ion-pairing (RPIP)-HPLC for detection of different types of GAGs in multiple species (Ledin et al., 2004; Lawrence et al., 2008; Yamada et al., 2011; Holmborn et al., 2012).

C. elegans synthesizes HS with modifications similar to those found in mammals and other organisms, but like other ecdysozoa the nematodes do not produce hyaluronan (Yamada et al., 1999; Toyoda et al., 2000; Lawrence et al., 2008; Townley and Bulow, 2011). However, although vast amounts of non-sulfated chondroitin were detected, CS was not previously identified, giving C. elegans a unique position among the ecdysozoa (Yamada et al., 1999; Toyoda et al., 2000).

In our protocol we separated sulfated and non-sulfated GAGs prior to analysis, facilitating detection of CS occurring in much lower quantities than the non-sulfated chondroitin (Dierker et al., 2016). Here, this method is described in detail.

Materials and Reagents

  1. Pipette tips (SARSTEDT, catalog numbers: 70.1114.100 , 70.760.502 , 70.762.200 )
  2. Tissue paper
  3. 15 ml tubes (SARSTEDT, catalog number: 62.554.502 )
  4. 50 ml tubes (SARSTEDT, catalog number: 62.547.254 )
  5. 1.5 ml Screwlock tubes (SARSTEDT, catalog number: 72.692.100 )
  6. Syringes 1 ml (Terumo Medical, catalog number: SS+01T1 )
  7. Syringes 2 ml (BD, catalog number: 300185 )
  8. MicrolanceTM 3 18 G needles (VWR, catalog number: 613-3945 )
  9. MicrolanceTM 3 23 G needles (VWR, catalog number: 613-3923 )
  10. MicrolanceTM 3 27 G needles (VWR, catalog number: 613-3834 )
  11. 10 ml round-bottom tubes
  12. Parafilm
  13. 0.45 µm filters (for example EMD Millipore, catalog number: HAWP04700 )
  14. Petri dishes 10 cm (SARSTEDT, catalog number: 82.1473.001 )
  15. 10 ml Poly-Prep® Chromatography columns (Bio-Rad Laboratories, catalog number: 7311550 )
  16. DEAE Sephacel (GE Healthcare, catalog number: 17050001 )
  17. 1.5 ml reaction tubes (SARSTEDT, catalog number: 72.690.001 )
  18. 2 ml reaction tubes (SARSTEDT, catalog number: 72.695.500 )
  19. NAP-10 columns (GE Healthcare, catalog number: 17-0854-02 )
  20. HPLC tubes (VWR, catalog number: 548-0440 )
  21. Locks (Fisher Scientific, catalog number: 11521434 )
    Note: This product has been discontinued.
  22. C. elegans and bacterial strains
    Note: All C. elegans strains as well as E. coli strain HB101 were obtained from the Caenorhabditis Genetics Center (CGC) https://cbs.umn.edu/cgc/home.
  23. Sodium hypochlorite (NaClO 6-14% active Cl) (Sigma-Aldrich, catalog number: 13440 )
  24. Protease XIV (Sigma-Aldrich, catalog number: P5147-5G )
  25. Benzonase (Merck, catalog number: 70746-3 )
  26. Heparin lyase I, II, III (IBEX Pharmaceuticals, catalog numbers: 50-010 , 50-011 , 50-012 )
  27. Quant-iTTM Broad Range Assay Kit (purchased from Molecular Probes)
  28. Calcium chloride (CaCl2·2H2O) (Merck, catalog number: 1.02382.0500 )
  29. Potassium hypochlorite (KOH) (Sigma-Aldrich, catalog number: P1767 )
  30. Potassium hydroxide (KOH) (Merck, catalog number: 1.05021.0250 )
  31. Magnesium sulfate heptahydrate (MgSO4·7H2O) (Merck, catalog number: 1.05886 )
  32. Cholesterol (Sigma-Aldrich, catalog number: C8667 )
  33. Ethanol (SOLVECO, catalog number: 1015 )
  34. Potassium dihydrogen phosphate (KH2PO4) (Merck, catalog number: 1.04873.1000 )
  35. di-Potassium hydrogen phosphate trihydrate (K2HPO4·3H2O) (Merck, catalog number: A257899 )
    Note: This product has been discontinued.
  36. 1 M K2HPO4
  37. Sodium chloride (NaCl) (Riedel-de Haën, catalog number: 31439 )
  38. Agarose (Sigma-Aldrich, catalog number: A9539 )
  39. Bacto peptone (BD, BactoTM, catalog number: 211677 )
  40. Bacteriological agar (VWR, catalog number: 84609.0500 )
  41. LB broth 1.1 G per tablet (Sigma-Aldrich, catalog number: L7275-500TAB )
  42. Bacto yeast extract (BD, BactoTM, catalog number: 212750 )
  43. di-Sodium hydrogen phosphate dodecahydrate (Na2HPO4·12H2O) (Merck, catalog number: 1.06579.100 )
  44. Magnesium chloride hexahydrate (MgCl2·6H2O) (Sigma-Aldrich, catalog number: M2670-500G )
  45. Triton X-100 (Fisher Scientific, catalog number: BP151-500 )
  46. Tris ultrapure (AppliChem, catalog number: A1086.1000 )
  47. 6 N HCl
  48. Sodium acetate (NaOAc) (Merck, catalog number: 1.06268.1000 )
  49. Silver nitrate (AgNO3) (Sigma-Aldrich, catalog number: S6506-5G )
  50. Acetic acid (Merck, catalog number: 1.00063.2500 )
  51. HEPES (Sigma-Aldrich, catalog number: H4034 )
  52. Chondroitinase ABC (Amsbio, catalog number: AMS.E1028-10 ; Sigma-Aldrich, catalog number: C3667 )
  53. Media for C. elegans growth and handling (see Recipes)
    1. 1 M CaCl2
    2. 5 N KOH
    3. 1 M MgSO4
    4. Cholesterol solution
    5. 1 M potassium phosphate buffer pH 6.0 (KPO4)
    6. Rich Nematode Growth medium (RNGM)
    7. LB agar
    8. 2x YT medium
    9. M9 buffer
  54. Buffers for GAG purification (see Recipes)
    1. 20% ethanol
    2. 1 M MgCl2
    3. Stock solutions
      i. 10% Triton X-100
      ii. 5 M NaCl
      iii. 1 M CaCl2
      iv. 1 M Tris-HCl
    4. Protease buffer
    5. DEAE elution buffer
    6. DEAE wash buffers
    7. DEAE wash buffer, pH 8
    8. DEAE wash buffer, pH 4
    9. 0.1% (w/v) AgNO3
    10. 5x chondroitinase ABC buffer
    11. 2x heparinase lyase buffer

Equipment

  1. Pipettes (for example Eppendorf, model: Research® plus )
  2. 125 ml Erlenmeyer flask (autoclaved)
  3. Rubber bulb
  4. 10 ml glass pipettes (autoclaved, plugged)
  5. Centrifuge
  6. Incubator for C. elegans (Lovibond Thermostatic Cabinet)
  7. Stereo microscope (Nikon Instruments, model: SMZ745 , eyelens: C-W10xB/22, magnification 2.5x to 50x)
  8. Hybridization oven (Hybaid Shake ‘n’ Stack)
  9. 37 °C incubator (Heraeus Instruments)
  10. SpeedVac (Labconco Freezer Dry System connected to Savant SpeedVac concentrator or Savant Instruments, model: SC11OA SpeedVac® Plus )
  11. HPLC system
    1. Merck Hitachi FL Detector L-7485 (Hitachi, model: Model L-7485 )
    2. Interface D-7000 (Hitachi, model: Model D-7000 )
    3. Autosampler L-7200 (Hitachi, model: Model L-7200 )
    4. Pump L-7100 (Hitachi, model: Model L-7100 )
    5. Column Oven L-7350 (Hitachi, model: Model L-7350 )
    6. Reagent pumps: Shimadzu LC-10AD (Shimadzu, model: LC-10AD ); Column: Phenomenex Luna 5u C18(2) 100A (Shimadzu)

Software

  1. D-7000 HPLC System Manager (HSM) software version 4.1
  2. GraphPad Prism version 5.0b for Mac OS X (GraphPad Software, San Diego California USA, www.graphpad.com

Procedure

A flowchart of the complete procedure is shown in Figure 1.


Figure 1. Flowchart of C. elegans growth and GAG purification

Note: Unless mentioned otherwise, all work was performed on a lab bench at room temperature under non-sterile conditions. Handling of C. elegans plates was performed close to an ethanol burner and all pipettes, pipette tips and bottles were quickly heat-sterilized by contact with the ethanol flame. All media and buffer compositions are described under ‘Recipes’ and are not mentioned in the text.

  1. Growth of C. elegans for GAG analysis
    To get large amounts of material for GAG analysis, worms were grown on rich nematode growth medium (RNGM) plates (see Recipes) (10-20 plates, 10 cm diameter) seeded with E. coli HB101. E. coli HB101 were chosen because they are easy for the worms to digest and allow recovery of many worms from the plates:
    1. Streak out HB101 bacteria from a glycerol stock on a fresh LB plate (see Recipes). Grow the bacteria over night at 37 °C.
    2. Inoculate 50 ml 2x YT medium (see Recipes) with one bacterial colony. Shake overnight (app. 16 h) at 200 rpm in a 125 ml Erlenmeyer flask at 37 °C.
    3. Prepare 10 cm RNGM plates. Let them dry overnight.
    4. Next day: Seed plates with a continuous layer (app. 600 µl of bacterial culture, outlined in black in Figure 2) of HB101. Dry the plates overnight on the bench and store until needed at 4 °C in a sealed container containing tissue paper.


      Figure 2. Outline of a continuous bacterial layer

    5. Transfer mixed stages of C. elegans to RNGM plates (10 plates per strain for RPIP-HPLC, 20 plates for LC-MS/MS or experiments that require multiple down-stream processing steps) by cutting chunks of C. elegans maintenance plates (prepared according to procedures described here: http://www.wormbook.org/chapters/www_strainmaintain/strainmaintain.html#d0e164), which are placed facedown onto the RNGM plates.
    6. Grow the animals until the plates contain many worms but make sure that they are not yet starved (i.e., confirm that the plates still have plenty of bacterial food, and that they do not have many dauers, developmentally arrested animals that are often seen under harsh growth conditions, like starvation, heat stress and high population density, etc.). Depending on the strain and the number of transferred animals this normally takes three to seven days.
    7. Separate plates inoculated with the same strain (10-20 plates, depending on experiment) into piles of five. Collect the worms by pipetting 10 ml M9 buffer (see Recipes) onto the first plate (use a rubber bulb and 10 ml autoclaved glass pipettes). Rinse the plate three times with the same solution and detach worms mechanically by carefully scraping the agar surface with the pipette after the second rinse. Pipette the same 10 ml onto the second plate and repeat until the five plates all have been washed with the same buffer solution. Repeat the procedure with 10 ml of fresh M9 buffer, this time starting with the fifth plate, followed by the fourth until all five plates have been washed twice.
    8. Pool all washing fractions (in 15 or 50 ml tubes, depending on the sample volume) and collect worms by centrifugation for 4 min at 500 x g.
    9. Remove the supernatant carefully (pipette and rubber bulb) and disperse the worm pellet in 5-10 ml M9 buffer (depending on bacterial density in buffer and sample volume). Repeat centrifugation and washing until no traces of bacteria are visible (normally two to three washing steps are required).
    10. Remove M9 buffer from the worm pellet and wash with 5-10 ml ddH2O in order to decrease the remaining amount of salt. Repeat centrifugation as above and transfer pelleted worms (normally between 100 and 500 µl) to a 1.5 ml screw-lock tube.
      Note: Cut off the tip of the 1 ml pipette tip to be used for transferring the worm pellet.
    11. Centrifuge as above and remove as much supernatant as possible. Rotate tubes in SpeedVac, apply vacuum (make sure that vacuum is establishd within a few minutes) and dry samples overnight.
      Note: If dry weight needs to be determined, the weight of empty tubes should be determined before the worms are added. The difference in weight between the tubes containing the dried worms and empty tubes represents the dry weight of the worm cultures.
    Notes:
    1. HB101 cultures can be stored at 4 °C for up to two weeks.
    2. Seeded RNGM plates were usually prepared when needed and should not be stored for longer than two weeks.
    3. When many RNGM plates are inoculated in parallel, it is important to choose agar chunks that contain approximately the same number of worms, so that all plates will be full of worms at the same time.
    4. When washing RNGM plates with M9, it is important to reverse the order of plates for the second wash to ensure that all plates are washed with sufficiently clean M9 buffer as bacteria are also washed off.
    5. If many worms are still present on the plates after two washes, use another 10 ml M9 to collect the residual animals of the same genotype/treatment from up to 10 RNGM plates.
    6. Dried C. elegans samples can be stored at -20 °C for months without affecting GAG isolation.

  2. Isolation of C. elegans eggs for GAG analysis
    When GAG analysis was performed on C. elegans embryonic stages, worms were grown as mixed stages (i.e., non-synchronized, thus containing embryonic, larval and adult stages, see http://wormatlas.org/hermaphrodite/introduction/Introframeset.html) on RNGM plates as described above and collected in M9 buffer as described.
    Note: The third washing step described in Note e of section A is strongly recommended since eggs tend to stick to the plates.
    1. Pellet worms by centrifugation at 4 °C for 4 min at 2,000 x g and remove the supernatant with a pipette.
    2. Incubate worm pellets with 3.5 ml ddH2O, 1 ml NaClO and 0.5 ml 5 N KOH until everything but worm eggs is dissolved (approximately 8 min).
      Note: Keep the tubes in your hand to increase temperature and rotate them constantly or even mix vigorously. Check the progress of dissolving by regularly inspecting the tubes under a stereo-microscope in order to avoid over-bleaching and killing of the eggs.
    3. Pellet worm eggs by centrifugation at 4 °C for 2 min at 1,300 x g. Remove the supernatant and wash the eggs with 5 ml ddH2O followed by centrifugation at 4 °C for 1 min at 2,000 x g. Repeat the washing step twice.
      Note: These conditions might be too harsh to revive larvae from the eggs as they are optimized for collecting eggs that would be homogenized soon after.
    4. Transfer eggs to a screw-lock tube, centrifuge as above and dry under vacuum as described in step A11.

  3. Sample lysis
    Sample lysis and purification of GAGs using anion exchange chromatography were adapted from previously described methods (Ledin et al., 2004; Dagalv et al., 2011). If samples had been frozen, they were briefly thawed at room temperature (up to 15 min).
    1. Add 80% of the required final volume (1 ml protease buffer [see Recipes] per 30 mg dry material) and boil the samples for 10 min at 96-100 °C, followed by a brief centrifugation to collect all liquid at the bottom of the tube.
    2. Pass samples consecutively through needles of decreasing diameters (see Materials and Methods). Use 1 or 2 ml syringes depending on the sample volume. For mixed stages of worms, start lysis with an 18 G needle (10 times passing up and down, carefully avoiding any overload or blockage of the needle), followed by a 23 G needle (same procedure as for 18 G). For worm eggs, it is sufficient to start with a 23 G needle. If possible, pass samples also through a 27 G needle.
      Note: 27 G needles tend to clog quickly so use with great care! Use the same syringe for all steps and empty needles as much as possible to avoid loss of material.
    3. Weigh Protease type XIV powder and dissolve in protease buffer to give a final concentration of 0.8 mg/ml. For example, if the final volume is 1 ml, 0.8 mg Protease type XIV is dissolved in 0.2 ml protease buffer and added to the 0.8 ml of homogenized sample.
    4. Incubate samples at 55 °C for around 24 h with constant rotation in a hybridization oven.
      Note: Screw-lock tubes are essential at this step as regular reaction tubes might not remain tight.
    5. Heat-inactivate the protease by a 10 min incubation at 96-100 °C. Centrifuge the samples for 10 min at maximum speed (16,060 x g).
    6. If DNA concentration will be determined, keep 4 µl of each sample for this purpose.
      Note: Can be performed immediately or after storage at -20 °C.
    7. Add 1 M MgCl2 to a final concentration of 2 mM MgCl2 and add 38 U (one unit digests 37 µg DNA in 30 min) Benzonase per ml lysate. Incubate samples overnight (normally 16-18 h) at 37 °C.
      Note: If many samples are handled in parallel, a master mix of 1 M MgCl2 and Benzonase is useful.
    8. Inactivate Benzonase followed by centrifugation as described above for Protease inactivation.
    Note: Protease XIV powder and Benzonase solution are stored at -20 °C.

  4. Ion exchange chromatography for CS purification
    Non-sulfated chondroitin (Chn) is highly abundant in C. elegans and may obscure the isolation of sulfated GAGs. The protocol presented below has been optimized for separation of the sulfated GAGs HS and CS (Dierker et al., 2016). Details about incubation times and temperature can be found in Figure 3 and the salt concentration of the buffers used during the different steps is shown in Figure 4.


    Figure 3. Incubation times and incubation temperature for ion exchange chromatography


    Figure 4. NaCl concentration in high affinity and low affinity GAG-fractions

    1. Transfer sufficient DEAE-Sephacel slurry to 10 ml Poly-Prep® Chromatography columns for bed volumes of 600 µl (Ledin et al., 2004) (see Figure 5).
      Note: Slurry can be transferred with a spatula, by pipetting or by pouring it into the column. To maintain the resin in the best state for long-term storage, it is recommended that the slurry is mixed well and that plastic pipette tips are cut off before pipetting.


      Figure 5. Final bed volume of DEAE columns

    2. Wash the columns with 2 ml (> three column volumes) 1.5 M NaCl, ddH2O and DEAE wash buffer pH 8 (see Recipes), respectively, before loading the sample.
      Note: Columns can be prepared in advance but should then be rehydrated before use.
    3. Adjust the final concentration of NaCl in the lysate to 0.25 M using 5 M NaCl.
    4. Load the lysates to the pre-washed columns (Note: Avoid any precipitate since this might clog the column–repeat centrifugation described in step C5 if samples have been frozen!). Seal the columns with the provided caps (top) and locks (bottom) as shown in Figure 6. Incubate for 1 h at 4 °C on an orbital rocking table (see Figures 2 and 7).


      Figure 6. Sealed DEAE column with sample loaded


      Figure 7. Sealed column incubated on a rocking table

    5. Remove caps and locks and collect the flow through fractions in 15 ml reaction tubes. Dilute the flow through fraction with ddH2O to give a final concentration of 0.1 M NaCl before loading it to a second column (prepared in parallel with the first one). Incubate as described in step D4 for 1 h.
      Note: GAGs binding to the first column, are designated ‘high affinity’ fraction and GAGs binding to the second column are defined as ‘low affinity’ fraction.
    6. Remove caps and locks and allow the samples to pass through columns.
    7. Wash all columns with 4 ml DEAE wash buffer pH 8, seal as before and incubate the columns for 10-15 min on a rocking plate at room temperature.
    8. Remove caps and locks and allow the wash buffer to pass through the columns (see Figure 8), followed by washing with 4 ml DEAE wash buffer pH 4 (see Recipes). Seal as before and incubate for 10-15 min on a rocking plate at room temperature.


      Figure 8. Allowing the wash buffer to pass through the column

    9. Remove caps and locks and allow the wash buffer to pass through the columns, followed by washing with 3 ml 0.1 M NaCl (low affinity) or 0.25 M NaCl (high affinity). Seal as before and incubate for 15-30 min on a rocking plate at room temperature.
    10. Remove caps and locks and allow the wash buffer to pass through the columns. Allow the DEAE-resin to drain completely and add 5.4 ml 1.5 M NaCl per column. Seal and incubate on a rocking plate at room temperature for 1 h in order to elute GAGs efficiently.
    11. Collect eluates in 10 ml round-bottom tubes. Reduce the volumes of the eluates by SpeedVac centrifugation to approximately 1.7 ml.
    Notes:
    1. Ensure that the bottom of the columns is sealed before applying the new buffer.
    2. Parafilm can be used to seal the columns, but the provided locks are very tight and easier to handle.
    3. Overloading the DEAE columns can reduce recovery. We calculated the required bed volume based on the binding capacity of DEAE for highly sulfated heparin to avoid overloading. However, these calculations might need to be adapted for other samples.

  5. Desalting of samples
    Desalting was performed using NAP-10 columns:
    1. Wash columns with at least six columns volumes of ddH2O, according to the manufacturer’s instructions.
    2. Determine the volume of the concentrated eluate and load it.
      Note: No more than 0.9 ml of sample can be desalted at a time, so load half of the eluate if the volume is more than that.
    3. Add ddH2O after the sample has entered the column (total volume of steps E2 and E3 should be 1 ml).
    4. Elute GAGs in 1.0-1.3 ml ddH2O (1.4 ml minus the volume used in step E3) into 10 ml round-bottom tubes.
    5. Wash columns with at least seven column volumes of ddH2O in order to prepare them for the second half of the eluate and repeat desalting procedure.
    6. Dry samples completely in a SpeedVac centrifuge.
    Notes:
    1. NAP-10 columns can be reused. Wash columns with at least seven columns volumes of ddH2O and store them in 20% EtOH at room temperature.
    2. In order to avoid contamination between samples, columns can also be washed with 1 ml 5 M NaCl followed by exhaustive washing with ddH2O in order to remove any remaining high molecular weight contaminants.
    3. Dried GAGs normally have a ‘fluffy’, white appearance while residual salt is white, and crystalline. Dissolve samples that are suspected to contain salt in 100 µl ddH2O and mix 1 µl of the sample with a drop of 0.1% AgNO3. If a white cloud appears in the solution (similar to or stronger than in a parallel tube with 1 µl 0.1 M NaCl) dissolve sample in 500 µl ddH2O and repeat desalting. Perform this step for all parallel samples to ensure that they are treated in the same way.

  6. GAG degradation and HS purification
    The heparinases used by us also show a weak CS-degrading activity, which is why it is important to completely remove CS prior to HS digestion and analysis:
    1. Reconstitute desalted samples in ddH2O.
    2. Add 20 µl of 5x chondroitinase buffer (final volume: 100 µl) (see Recipes) per tube.
    3. Add approximately 1 mU chondroitinase (CSase) ABC per mg dried starting material.
    4. Incubate overnight (minimum 16 h) at 37 °C.
    5. Heat-inactivate CSase ABC at 96-100 °C for 10 min and centrifuge the samples for 10 min at maximum speed.
    6. If HS analysis is not carried out, transfer the complete samples to HPLC tubes and adjust volumes to 100 µl with ddH2O to compensate for any loss during the incubation time. Perform disaccharide analysis as described under ‘Data analysis’.
    7. If in addition information about the HS composition will be determined, save 5-20% of the sample to an HPLC tube for CS compositional analysis.
    8. Adjust the volume of the remaining sample to 500 µl with ddH2O and 5 M NaCl to achieve a final concentration of 0.1 M NaCl.
    9. Prepare DEAE columns (200 µl bed volume) as described under Procedure D.
    10. Load samples, seal columns and incubate for 1 h at 4 °C while rocking.
    11. Wash the columns with 1.2 ml DEAE wash buffer pH 8, 1.2 ml DEAE wash buffer pH 4 and 1 ml 0.1 M NaCl without further incubation.
    12. Let the wash buffer drain from columns and elute HS by incubating the columns with 1.8 ml 1.5 M NaCl for 1 h at room temperature while rocking.
    13. Collect eluates in 2 ml reaction tubes and concentrate them in a SpeedVac centrifuge to 600-800 µl.
    14. Desalt on NAP-10 columns as described under Procedure E.
      Note: The amount of HS in the samples is much lower than that of CS, so barely any precipitate is expected after drying.
    15. Reconstitute the desalted samples in 100 µl ddH2O, divide them into 2x 50 µl aliquots and add 49 µl of 2x HS digestion buffer to both of them.
    16. Add 1 µl heparinase mix (0.4 mU each of heparinase I, heparinase II, and heparinase III) to one aliquot and 1 µl 1x HS digestion buffer (see Recipes) to the other one.
      Note: The non-digested sample is used as a background control in the HPLC-run, see ‘Data analysis’).
    17. Incubate all samples overnight (more than 16 h) at 37 °C.
    18. Heat-inactivate, centrifuge and transfer the samples to HPLC tubes as described under steps F5 and F6.
    Notes:
    1. HS and CS digestion buffers can be aliquoted and stored at -20 °C to prolong shelf life.
    2. Other buffers are stored at room temperature.
    3. CSase ABC (-20 °C) and heparin lyases (-80 °C) are stored in aliquots of 5-20 µl and multiple freezing is avoided.

Data analysis

  1. Reversed-phase ion-pairing HPLC
    Conditions for disaccharide separation (generated by either CSase ABC digestion or by cleavage with a mixture of heparin lyase I, II and III) and post-column derivatisation with 2-cyanoacetamide were described by Ledin et al. (2004) and slightly modified by Dagalv et al. (2011).
    1. Use disaccharide standards analyzed in the same sample series to identify and quantify the peaks based on their elution position and peak size using the D-7000 HPLC System Manager (HSM) software version 4.1.
      Note: Prepare similar concentrations (µg/µl) of the different types of HS and CS disaccharides as stock solutions, and mix these to generate HS and CS standards. Add a multiplying factor to the D-7000 HSM software in order to account for the different molecular masses of the disaccharides.
    2. In our hands, background noise in the HS samples (absent from the CS samples) requires the non-enzyme treated sample to be used as background control; subtract this from the chromatogram of the enzyme treated sample.
    3. Run treated and non-treated samples directly following each other to minimize changes in buffer conditions between the runs.
    4. The D-7000 HSM software exports data into an Excel file. We recommend to use a ‘macro’ (set of programming instructions for Excel) to calculate the total pmol per peak based on the signal for the relevant standard. A chromatogram as well as the resulting calculations are shown in Figure 9.


      Figure 9. Chromatogram (upper panel) and calculations (lower panel) as exported by D-7000 HSM software

    5. Use total pmol values to calculate the relative amount of a certain disaccharide (disaccharide composition) in each sample.
    6. The total sulfation is calculated based on the relative amount of sulfated disaccharides, and is therefore given as sulfation per 100 disaccharides.
    7. In order to quantify the total recovery from a given sample the amount (in pmol) of GAG recovered can be normalized to mg dry weight, after correction for the proportion of injected sample.
      Note: In general, 10-90 µl were injected to ensure that the column was not overloaded.
    8. As an alternative to dry weight, samples can also be normalized to DNA content before Benzonase treatment (see Procedure C, step C7). We use Quant-iTTM Broad Range Assay Kit (purchased from Molecular Probes) according to the manufacturer’s instructions and are satisfied with the results using this method.
    9. When comparing wild type and mutant strains we use samples that are purified and analyzed in parallel. We consider samples grown and isolated on different days as independent experiments and normally conduct at least three independent experiments.

  2. Statistical analysis and data presentation
    1. All graphs are generated using GraphPad Prism version 5.0b for Mac OS X (GraphPad Software, San Diego California USA, www.graphpad.com).
    2. Diagrams show mean values and error bars define standard error of mean (SEM).
    3. One-tailed Mann-Whitney tests are performed where possible and P-values < 0.05 are considered significant.

Notes

  1. Growth conditions will affect GAG composition, most likely due to changes in GAG structures during C. elegans development. We therefore suggest to only compare worms that have been grown under similar conditions. Comparing animals of the same developmental stage strongly improved reproducibility in our hands.
  2. Even though NAP-10 columns can be reused and should not retain high molecular weight molecules, we suggest reusing columns only for similar samples and not for material from other species to avoid contamination.
  3. When analyzing the chain length of GAGs from C. elegans we noted that they appeared to be shorter than those from mammals. The protocol presented here might therefore need optimization for separation of sulfated and non-sulfated GAGs from other species.

Recipes

Note: Unless noted otherwise buffers were prepared with non-autoclaved, deionized water and stored at room temperature. Sterilized buffers were either autoclaved (120 °C, 20 min) or passed through a 0.45 µm filter.

  1. Media for C. elegans growth and handling
    1. 1 M CaCl2
      1.47 g CaCl2
      Adjust volume to 10 ml
    2. 5 N KOH
      14.028 g KOH
      Adjust volume to 50 ml
    3. 1 M MgSO4
      12.324 g MgSO4
      Adjust volume to 50 ml
    4. Cholesterol solution
      5 mg/ml in 99.5% ethanol
      Sterilize by filtration
    5. 1 M potassium phosphate buffer pH 6.0 (KPO4)
      120 g KH2PO4
      27.55 g K2HPO4·3H2O per 1 L
      Adjust pH to 6.0 with 1 M K2HPO4
      Autoclave
    6. Rich Nematode Growth medium (RNGM)
      1.5 g NaCl
      7.5 g agarose
      3.75 g Bacto peptone per 500 ml
      Autoclave
      Cool to less than 55 °C and then add:
      500 µl cholesterol
      500 µl 1 M MgSO4
      500 µl 1 M CaCl2
      12.5 ml 1 M KPO4 buffer
      Mix carefully before dispensing into Petri culture dishes of the desired size
      Dry plates at room temperature and store at 4 °C
    7. LB agar
      2.5 g bacteriological agar
      5 LB tablets per 250 ml
      Autoclave
      Store at 4 °C
    8. 2x YT medium
      1.6 g Bacto peptone
      1 g Bacto yeast extract
      0.5 g NaCl per 100 ml
      Autoclave
      Store at 4 °C
    9. M9 buffer
      5 g NaCl
      15.12 g Na2HPO4·7H2O
      3 g KH2PO4 per 1 L
      Autoclave
      Add 1 ml 1 M MgSO4

  2. Buffers for GAG purification
    Note: All buffers for washing and elution from DEAE columns were filtered through 0.45 µm filters.
    1. 20% ethanol
      100 ml 99% ethanol
      Adjust volume to 500 ml
    2. 1 M MgCl2
      2.03 g MgCl2
      Adjust volume to 10 ml
    3. Stock solutions
      1. 10% Triton X-100
        5 ml Triton X-100 (careful, very viscous!)
        Adjust volume to 50 ml
      2. 5 M NaCl
        292.2 g NaCl
        Adjust volume to 1 L
        Note: Filtering the 5 M NaCl is an alternative to filtering all DEAE buffers.
      3. 1 M CaCl2 (see Recipes A1)
      4. 1 M Tris-HCl
        121.14 g Tris
        Adjust pH to 7.5 with 6 N HCl
        Adjust volume to 1 L
        Autoclave
    4. Protease buffer (50 mM Tris/HCl pH 8.0, 1 mM CaCl2, 1% Triton X-100)
      5 ml 1 M Tris-HCl
      100 µl 1 M CaCl2
      10 ml 10% Triton X-100 on 80 ml ddH2O
      Adjust pH with 6 M HCl
      Adjust volume to 100 ml
      Store at 4 °C
    5. DEAE elution buffer (1.5 M NaCl)
    6. DEAE wash buffers (0.1 M NaCl or 0.25 M NaCl)
    7. DEAE wash buffer, pH 8 (50 M Tris/HCl pH 8.0, 0.1 M NaCl)
      25 ml 1 M Tris-HCl
      10 ml 5 M NaCl on 400 ml ddH2O
      Adjust pH with 6 M HCl
      Adjust volume to 500 ml
    8. DEAE wash buffer, pH 4 (50 mM NaOAc pH 4.0, 0.1 M NaCl)
      25 ml 1 M NaOAc
      10 ml 5 M NaCl on 400 ml ddH2O
      Adjust pH with 1 M acetic acid
      Adjust volume to 500 ml
    9. 0.1% AgNO3
      100 mg AgNO3
      Adjust volume to 100 ml
    10. 5x chondroitinase ABC buffer (0.2 M Tris-acetate pH 8.0)
      1.211 g Tris in 40 ml ddH2O
      Adjust pH with 1 M acetic acid
      Adjust volume to 50 ml
    11. 2x heparinase lyase buffer (10 mM HEPES pH 7.0, 2 mM CaCl2)
      119.2 mg HEPES
      100 µl 1 M CaCl2 on 40 ml ddH2O
      Adjust pH with 6 M HCl
      Adjust volume to 50 ml

Acknowledgments

Funding from the Swedish Research Council (to L.K.), the German Academic Exchange Service (to T.D.) and Stiftelsen för Proteoglykanforskning (to L.K.) are acknowledged. GAG purification was described by Ledin et al., JBC 2004 (doi: 10.1074/jbc.M405382200) and the protocol modified for C. elegans was first published by Dierker et al., Sci. Rep. 2016 (doi: 10.1038/srep34662).

References

  1. Bishop, J. R., Schuksz, M., and Esko, J. D. (2007). Heparan sulphate proteoglycans fine-tune mammalian physiology. Nature 446(7139): 1030-1037.
  2. Bulow, H. E., and Hobert, O. (2006). The molecular diversity of glycosaminoglycans shapes animal development. Annu Rev Cell Dev Biol 22: 375-407.
  3. Dagalv, A., Holmborn, K., Kjellen, L., and Abrink, M. (2011). Lowered expression of heparan sulfate/heparin biosynthesis enzyme N-deacetylase/n-sulfotransferase 1 results in increased sulfation of mast cell heparin. J Biol Chem 286(52): 44433-44440.
  4. Dierker, T., Shao, C., Haitina, T., Zaia, J., Hinas, A., and Kjellen, L. (2016). Nematodes join the family of chondroitin sulfate-synthesizing organisms: Identification of an active chondroitin sulfotransferase in Caenorhabditis elegans. Sci Rep 6: 34662.
  5. Holmborn, K., Habicher, J., Kasza, Z., Eriksson, A. S., Filipek-Gorniok, B., Gopal, S., Couchman, J. R., Ahlberg, P. E., Wiweger, M., Spillmann, D., Kreuger, J., and Ledin, J. (2012). On the roles and regulation of chondroitin sulfate and heparan sulfate in zebrafish pharyngeal cartilage morphogenesis. J Biol Chem 287: 33905-33916.
  6. Kiselova, N., Dierker, T., Spillmann, D., and Ramstrom, M. (2014). An automated mass spectrometry-based screening method for analysis of sulfated glycosaminoglycans. Biochem Biophys Res Commun 450: 598-603.
  7. Lawrence, R., Olson, S. K., Steele, R. E., Wang, L., Warrior, R., Cummings, R. D., and Esko, J. D. (2008). Evolutionary differences in glycosaminoglycan fine structure detected by quantitative glycan reductive isotope labeling. J Biol Chem 283(48): 33674-33684.
  8. Ledin, J., Staatz, W., Li, J. P., Götte, M., Selleck, S., Kjellén, L., and Spillmann, D. (2004). Heparan sulfate structure in mice with genetically modified heparan sulfate production. J Biol Chem 279(41): 42732-42741.
  9. Shao, C., Shi, X., Phillips, J. J., and Zaia, J. (2013). Mass spectral profiling of glycasaminoglycans from histological tissue surfaces. Anal Chem 85(22): 10984-10991.
  10. Townley, R. A. and Bulow, H. E. (2011). Genetic analysis of the heparan modification network in Caenorhabditis elegans. J Biol Chem 286:16824-16831.
  11. Toyoda, H., Kinoshita-Toyoda, A., and Selleck, S. B. (2000). Structural analysis of glycosaminoglycans in Drosophila and Caenorhabditis elegans and demonstration that tout-velu, a Drosophila gene related to EXT tumor suppressors, affects heparan sulfate in vivo. J Bioll Chem 275(4): 2269-2275.
  12. Yamada, S., Sugahara, K., and Ozbek, S. (2011). Evolution of glycosaminoglycans. Commun Integr Biol 4(2): 150-158.
  13. Yamada, S., Van Die, I., Van den Eijnden, D. H., Yokota, A., Kitagawa, H., and Sugahara, K. (1999). Demonstration of glycosaminoglycans in Caenorhabditis elegans. FEBS Lett 459(3): 327-331.
  14. Zhang, L. (2010). Progress in molecular biology and translational science. Elsevier 93: 1-17.

简介

线虫秀丽隐杆线虫是研究发育生物学,神经学,衰老等基础研究领域的流行模型生物。 因为许多发育过程由细胞表面和细胞外基质中的糖胺聚糖(GAG)调节,分离和分析C的方法。 线虫需要GAG。 此类方法先前已针对其他物种如小鼠和斑马鱼进行了优化。 在修改现有的纯化方案后,我们最近可以显示除了硫酸乙酰肝素外,线虫也产生硫酸软骨素,因此挑战了仅通过C合成非硫酸软骨素的观点。线虫。 我们在这里介绍我们适用于C的协议。线虫。 由于净化策略涉及非硫酸化和硫酸化GAG的分离,所以对于其他可能有利的方法也是有用的。
【背景】糖胺聚糖(GAG)是重复二糖单元的直链多糖链,其通常被硫酸根取代。除了透明质酸,其是在质膜上合成而不被锚定到任何蛋白质的非硫酸化GAG,所有其他GAG都与核心蛋白共价连接,从而形成蛋白聚糖(PG)。在PG上发现的最常见的GAG是硫酸乙酰肝素(HS)和硫酸软骨素(CS)/硫酸皮肤素,含有N-乙酰基 - 葡糖胺和N,N-乙酰基 - 半乳糖胺,分别(张,2010)。在高尔基隔室的生物合成过程中,它们是非模板驱动的过程,它们经过多种修饰,包括将葡萄糖醛酸差向异构化成艾杜糖醛酸,并在不同位置硫酸化(Bulow和Hobert,2006; Zhang,2010)。所产生的硫酸化模式对于GAG链与生长因子和细胞因子相互作用的能力是重要的,而生长因子和细胞因子又对其影响发育和其它重要生理过程的能力至关重要(Bishop等人, 2007)。
为了分析其组成,GAG需要从粗细胞或组织裂解液中纯化,最常见的是通过蛋白酶和核酸酶消化后的离子交换层析(Ledin等人,2004)来实现。然后可以使用不同的色谱方法或质谱法(Shao等人,2013; Kiselova等人,2014年)鉴定通过特异性GAG裂解酶的作用产生的二糖)。我们和其他几个实验室已经使用反相离子配对(RPIP)-HPLC来检测多种不同类型的GAG(Ledin等人,2004; Lawrence等人)。 ,2008; Yamada等人,2011; Holmborn等人,2012)。
℃。线虫合成具有类似于在哺乳动物和其他生物体中发现的修饰的HS,但是像其他ecdysozoa一样,线虫不产生透明质酸(Yamada等人,1999; Toyoda等人,2000; Lawrence等人,2008; Townley和Bulow,2011)。然而,虽然检测到大量的非硫酸软骨素,但是以前没有发现CS,给出了C。 elegans 在ecdysozoa(Yamada等人,1999; Toyoda等人,2000)中独特的地位。
在我们的方案中,我们在分析之前分离了硫酸化和非硫酸化的GAG,促进了以比非硫酸化软骨素低得多的量发现CS的发现(Dierker et al。,2016)。这里详细说明该方法。

关键字:糖胺聚糖, 秀丽隐杆线虫, 蛋白多糖, 离子交换色谱法, 硫酸化

材料和试剂

  1. 移液器吸头(SARSTEDT,目录号:70.1114.100,70.760.502,70.762.200)
  2. 组织纸
  3. 15毫升管(SARSTEDT,目录号:62.554.502)
  4. 50ml管(SARSTEDT,目录号:62.547.254)
  5. 1.5ml螺丝锁管(SARSTEDT,目录号:72.692.100)
  6. 注射器1毫升(Terumo医疗,目录号:SS + 01T1)
  7. 注射器2 ml(BD,目录号:300185)
  8. Microlance TM 3 18 G针(VWR,目录号:613-3945)
  9. Microlance TM 3/3 3 G针(VWR,目录号:613-3923)
  10. Microlance TM 3/2 3 G针(VWR,目录号:613-3834)
  11. 10毫升圆底管
  12. 石蜡膜
  13. 0.45μm过滤器(例如EMD Millipore,目录号:HAWP04700)
  14. 培养皿10厘米(SARSTEDT,目录号:82.1473.001)
  15. 色谱柱(Bio-Rad Laboratories,目录号:7311550)
  16. DEAE Sephacel(GE Healthcare,目录号:17050001)
  17. 1.5ml反应管(SARSTEDT,目录号:72.690.001)
  18. 2ml反应管(SARSTEDT,目录号:72.695.500)
  19. NAP-10色谱柱(GE Healthcare,目录号:17-0854-02)
  20. HPLC管(VWR,目录号:548-0440)
  21. 锁(Fisher Scientific,目录号:11521434)
    注意:本产品已停产。
  22. ℃。线虫和细菌株
    注意:所有秀丽隐杆线虫菌株以及大肠杆菌菌株HB101均从Caenorhabditis遗传学中心(CGC)获得 https://cbs.umn.edu/cgc/home
  23. 次氯酸钠(NaClO 6-14%活性Cl)(Sigma-Aldrich,目录号:13440)
  24. 蛋白酶XIV(Sigma-Aldrich,目录号:P5147-5G)
  25. Benzonase(默克,目录号:70746-3)
  26. 肝素裂解酶I,II,III(IBEX Pharmaceuticals,目录号:50-010,50-011,50-012)
  27. Quant-iT TM 宽范围测定试剂盒(从Molecular Probes购买)
  28. 氯化钙(CaCl 2·2H 2 O)(Merck,目录号:1.02382.0500)
  29. 次氯酸钾(KOH)(Sigma-Aldrich,目录号:P1767)
  30. 氢氧化钾(KOH)(默克,目录号:1.05021.0250)
  31. 七水硫酸镁(MgSO 4·7H 2 O)(Merck,目录号:1.05886)
  32. 胆固醇(Sigma-Aldrich,目录号:C8667)
  33. 乙醇(SOLVECO,目录号:1015)
  34. 磷酸二氢钾(KH 2 PO 4)(Merck,目录号:1.04873.1000)
  35. 磷酸氢二钾三水合物(K 2 HPO 4·3H 2 O)(Merck,目录号:A257899)
    注意:本产品已停产。
  36. 1 M K 2 HPO 4
  37. 氯化钠(NaCl)(Riedel-deHaën,目录号:31439)
  38. 琼脂糖(Sigma-Aldrich,目录号:A9539)
  39. 细菌蛋白胨(BD,Bacto TM ,目录号:211677)
  40. 细菌琼脂(VWR,目录号:84609.0500)
  41. LB肉汤每片1.1G(Sigma-Aldrich,目录号:L7275-500TAB)
  42. 细菌酵母提取物(BD,Bacto TM,目录号:212750)
  43. 磷酸二氢钠十二水合物(Na 2 HPO 4·12H 2 O)(Merck,目录号:1.06579.100) >
  44. 氯化镁六水合物(MgCl 2·6H 2 O)(Sigma-Aldrich,目录号:M2670-500G)
  45. Triton X-100(Fisher Scientific,目录号:BP151-500)
  46. Tris Ultrapure(AppliChem,目录号:A1086.1000)
  47. 6 N HCl
  48. 乙酸钠(NaOAc)(Merck,目录号:1.06268.1000)
  49. 硝酸银(AgNO 3)(Sigma-Aldrich,目录号:S6506-5G)
  50. 乙酸(Merck,目录号:1.00063.2500)
  51. HEPES(Sigma-Aldrich,目录号:H4034)
  52. 软骨素酶ABC(Amsbio,目录号:AMS.E1028-10; Sigma-Aldrich,目录号:C3667)
  53. 生长和处理(见食谱)的C. elegans 的媒体
    1. 1 M CaCl 2
    2. 5 N KOH
    3. 1 M MgSO 4
    4. 胆固醇溶液
    5. 1M磷酸钾缓冲液pH 6.0(KPO 4
    6. 丰富的线虫生长培养基(RNGM)
    7. LB琼脂
    8. 2x YT媒体
    9. M9缓冲区
  54. GAG净化缓冲液(参见食谱)
    1. 20%乙醇
    2. 1 M MgCl 2
    3. 库存解决方案
      一世。 10%Triton X-100
      II。 5 M NaCl
      III。 1 M CaCl 2
      IV。 1 M Tris-HCl
    4. 蛋白酶缓冲液
    5. DEAE洗脱缓冲液
    6. DEAE洗涤缓冲液
    7. DEAE洗涤缓冲液,pH 8
    8. DEAE洗涤缓冲液,pH 4
    9. 0.1%(w / v)AgNO 3
    10. 5x软骨素酶ABC缓冲液
    11. 2x肝素酶裂合酶缓冲液

设备

  1. 移液器(例如Eppendorf,型号:Research ® plus)
  2. 125ml锥形瓶(高压灭菌)
  3. 橡胶灯泡
  4. 10毫升玻璃移液器(高压灭菌,堵塞)
  5. 离心机
  6. 孵化器为C.线虫(Lovibond Thermostatic Cabinet)
  7. 立体显微镜(Nikon Instruments,型号:SMZ745,眼线:C-W10xB / 22,放大倍率2.5x至50x)
  8. 杂交烤箱(Hybaid Shake'n'Stack)
  9. 37℃培养箱(Heraeus Instruments)
  10. SpeedVac(Labconco冷冻干燥系统连接到Savant SpeedVac集中器或Savant Instruments,型号:SC11OA SpeedVac ® Plus)
  11. HPLC系统
    1. 默克日立FL检测仪L-7485(日立,型号:L-7485型)
    2. 接口D-7000(日立,型号:D-7000)
    3. 自动进样器L-7200(日立,型号:L-7200)
    4. 泵L-7100(日立,型号:L-7100型)
    5. 立柱烤箱L-7350(日立,型号:L-7350)
    6. 试剂泵:Shimadzu LC-10AD(Shimadzu,型号:LC-10AD);柱:Phenomenex Luna 5u C18(2)100A(Shimadzu)

软件

  1. D-7000 HPLC系统管理器(HSM)软件版本4.1
  2. GraphPad Prism版本5.0b for Mac OS X(GraphPad Software,San Diego California USA, www.graphpad.com

程序

完整的流程图如图1所示

图1.线虫生长和GAG纯化的流程图

注意:除非另有说明,所有的工作都是在室温下在非无菌条件下在实验台上进行的。在乙醇燃烧器附近进行线虫板的处理,并且通过与乙醇火焰接触将所有移液管,移液管吸头和瓶迅速加热灭菌。所有介质和缓冲液组合物都在“配方”下进行描述,在文中未提及

  1. C的增长。线虫用于GAG分析
    为了获得大量的用于GAG分析的材料,蠕虫在富含线虫生长培养基(RNGM)板(参见食谱)(10-20平板,直径10cm)上种植。大肠杆菌HB101。 电子。 co li HB101被选中,因为它们易于蠕虫消化,并允许从板上恢复许多蠕虫:
    1. 从新鲜LB平板上的甘油储备液中分离出HB101细菌(参见食谱)。在37°C将细菌生长过夜。
    2. 接种50毫升2x YT培养基(见食谱)与一个细菌菌落。在37℃下,在125ml三角烧瓶中以200rpm摇动过夜(应用16小时)。
    3. 准备10厘米RNGM板。让他们一夜之间干
    4. 第二天:HB101的连续层的种子板(应用600μl的细菌培养物,在图2中以黑色概述)。将板子在板凳上干燥过夜,直到需要在4℃下储存在含有薄纸的密封容器中。


      图2.连续细菌层的概况

    5. 将线虫的混合阶段转移到RNGM板(每个菌株用于RPIP-HPLC的10个板,用于LC-MS / MS的20个板或需要多个下游处理步骤的实验),通过切割块℃。 elegans 维护板(根据这里描述的程序准备: http://www.wormbook.org/chapters/www_strainmaintain/strainmaintain.html#d0e164 ),这些面朝下放在RNGM板上。
    6. 生长动物直到盘子含有许多蠕虫,但确保它们还没有饿死( ie ,确认平板仍然有大量的细菌食物,他们没有很多的狗,被发现逮捕通常在恶劣的生长条件下看到的动物,如饥饿,热应激和高人口密度,等)。取决于通常需要三至七天的转移动物的菌株和数量。
    7. 用相同菌株(10-20个平板,取决于实验)接种的单独的板块成为5个堆。通过将10ml M9缓冲液(参见食谱)移至第一个板上(使用橡胶球和10 ml高压灭菌的玻璃移液管)收集蠕虫。用相同的溶液冲洗板三次,并在第二次冲洗后用移液管小心地刮去琼脂表面,机械地分开蠕虫。将相同的10ml移液到第二块板上,重复,直到五块板都用相同的缓冲溶液洗涤。用10ml新鲜的M9缓冲液重复此过程,此时从第五块板开始,然后第四次,直到所有的五个板都洗两次。
    8. 将所有洗涤组分(15或50 ml管,取决于样品体积)进行收集,并以500 x g离心4分钟收集蠕虫。
    9. 去除上清液(移液管和橡胶球),并将蠕虫颗粒分散在5-10 ml M9缓冲液中(取决于缓冲液中的细菌密度和样品体积)。重复离心和洗涤,直至没有细菌痕迹(通常需要2-3个洗涤步骤)。
    10. 从蠕虫颗粒中除去M9缓冲液,并用5-10ml ddH 2 O洗涤,以减少盐的剩余量。如上所述重复离心,并将沉淀的蠕虫(通常在100和500μl之间)转移到1.5 ml螺丝锁管。
      注意:切掉用于转移蠕虫颗粒的1毫升吸头的尖端。
    11. 如上所述离心,去除尽可能多的上清液。在SpeedVac中旋转管,应用真空(确保在几分钟内建立真空),并将样品干燥过夜。
      注意:如果需要确定干重,应在添加蠕虫之前确定空管的重量。含有干燥蠕虫和空管的管的重量差异代表蠕虫培养物的干重。

      注意:

      1. HB101培养物可以在4℃下储存长达两周。
      2. 种子RNGM板通常在需要时制备,不应该储存超过两周。
      3. 当许多RNGM平板并口接种时,重要的是选择含有大约相同数量蠕虫的琼脂块,以便所有的板都将同时充满蠕虫。
      4. 当使用M9洗涤RNGM板材时,重新进行第二次洗涤的板材的顺序,以确保所有的板材都用足够干净的M9缓冲液洗涤,同时也洗去细菌。
      5. 如果两次洗涤后,如果许多蠕虫仍然存在于板上,则使用另外的10ml M9从最多10个RNGM平板上收集相同基因型/治疗的残留动物。
      6. 干燥的线虫样品可以在-20℃下储存数月,不影响GAG分离。

  2. 用于GAG分析的线虫鸡蛋的分离
    当在C上进行GAG分析时。线虫胚胎阶段,蠕虫生长为混合阶段( ie ,不同步,因此包含胚胎,幼虫和成年阶段,见 http://wormatlas.org/hermaphrodite/introduction/Introframeset.html )上述RNGM板,并收集在M9缓冲液中。
    注意:强烈推荐第A部分注释e所述的第三次洗涤步骤,因为鸡蛋往往会粘在板上。
    1. 颗粒蠕虫通过在4℃下以2,000×g离心4分钟并用移液管除去上清液。
    2. 使用3.5ml ddH 2 O,1ml NaClO和0.5ml 5N KOH孵育蠕虫小球,直到除蠕虫卵溶解(约8分钟)为止。
      注意:将管子保持在手中以增加温度,并持续旋转甚至搅拌均匀。通过在立体显微镜下定期检查管子来检查溶解进程,以避免过度漂白和杀死鸡蛋。
    3. 通过在1,300xg下在4℃下离心2分钟来制备颗粒虫卵。去除上清液并用5ml ddH 2 O洗涤卵,然后在4℃下以2,000×g离心1分钟。重复洗涤步骤两次。
      注意:这些条件可能太苛刻,无法从卵中恢复幼虫,因为它们被优化用于收集即将到来的同质化鸡蛋。
    4. 将蛋转移到螺丝锁管,如上所述离心机,如步骤A11所述在真空下干燥。

  3. 样品溶解
    使用阴离子交换层析的样品裂解和纯化GAG由前述方法(Ledin等人,2004; Dagalv等人,2011)改编。如果样品已被冷冻,则在室温下短暂融化(最多15分钟)。
    1. 加入所需最终体积的80%(1ml蛋白酶缓冲液[参见食谱]每30mg干物质),并在96-100℃下煮沸样品10分钟,然后短暂离心以收集底部的所有液体管子。
    2. 通过直径减小的针连续通过样品(参见材料和方法)。根据样品量使用1或2 ml注射器。对于蠕虫的混合阶段,用18G针头开始裂解(10次通过上下,仔细避免任何过度或针头堵塞),然后用23G针头(与18G相同的程序)开始裂解。对于蠕虫卵,从23 G针开始就足够了。如果可能,也可以通过27 G针进行样品。
      注意:27 G针很容易堵塞,所以非常小心使用!尽可能使用相同的注射器进行所有步骤和空针,以避免材料损失。
    3. 称取蛋白酶XIV粉末,溶解在蛋白酶缓冲液中,得到最终浓度为0.8mg / ml。例如,如果最终体积为1ml,则将0.8mg蛋白酶XIV溶解于0.2ml蛋白酶缓冲液中并加入0.8ml均质样品中。
    4. 在杂交烘箱中,将样品在55℃孵育约24小时,恒定旋转。
      注意:螺旋锁管在此步骤中至关重要,因为常规反应管可能不会保持紧固。
    5. 在96-100℃温育10分钟,使蛋白酶热灭活。以最大速度离心样品10分钟(16,060 x g)。
    6. 如果确定DNA浓度,为了此目的,保留每个样品4μl 注意:可以立即执行或在-20°C储存后进行。
    7. 加入1M MgCl 2,终浓度为2mM MgCl 2,并加入38U(一个单元在30分钟内消化37μgDNA)每ml裂解物的Benzonase。在37℃下孵育样品过夜(通常为16-18小时)。
      注意:如果并行处理许多样品,则可以使用1 M MgCl 2 和Benzonase的主混合物。
    8. 停用苯甲酸酶,然后如上所述用于蛋白酶失活进行离心。

      注意:蛋白酶XIV粉末和Benzonase溶液储存在-20°C

  4. 离子交换色谱用于CS纯化
    非硫酸软骨素(Chn)在C中是非常丰富的。线虫,并且可能掩盖了硫酸化GAG的分离。以下提出的方案已经优化用于分离硫酸化GAG HS和CS(Dierker等人,2016)。关于孵育时间和温度的细节可以在图3中找到,并且在不同步骤期间使用的缓冲液的盐浓度如图4所示。


    图3.离子交换色谱的孵育时间和孵育温度


    图4.高亲和力和低亲和力GAG馏分中的NaCl浓度

    1. 将足够的DEAE-Sephacel浆液转移到10ml Poly-Prep(色谱柱)色谱柱中,床体积为600μl(Ledin等人,2004)(参见图5)。
      注意:泥浆可以用刮刀,移液或将其倒入色谱柱中进行转印。为了保持树脂处于最佳状态以进行长期储存,建议将浆料混合均匀,并在移液前将塑料移液器吸头切断。


      图5. DEAE列的最终床体积

    2. 在装载样品之前,分别用2ml(> 3柱体积)1.5M NaCl,ddH 2 O和DEAE洗涤缓冲液pH8洗涤柱子(参见食谱)。
      注意:可以预先准备色谱柱,然后在使用前再进行水化。
    3. 使用5 M NaCl将裂解液中NaCl的最终浓度调至0.25 M
    4. 将裂解物加载到预洗柱(注意:避免任何沉淀,因为如果样品已被冷冻,则可能会堵塞步骤C5中描述的柱重复离心!)。用提供的盖(顶部)和锁(底部)密封柱,如图6所示。在4°C的轨道摇床上孵育1小时(参见图2和图7)。


      图6.加载样品的密封DEAE列


      图7.在摇摆桌上孵育的密封柱

    5. 取出帽子和锁,并收集15ml反应管中的流分。用ddH 2 O稀释流过馏分,使其最终浓度为0.1M NaCl,然后将其装载到第二柱(与第一柱并行制备)。如步骤D4所述孵育1小时。
      注意:绑定到第一列的GAG被指定为“高亲和力”分数,并且与第二列结合的GAG被定义为“低亲和力”分数。
    6. 取出盖子和锁,并允许样品通过色谱柱。
    7. 用4ml DEAE洗涤缓冲液pH8洗涤所有柱,如前所述进行密封,并在室温下在摇床上孵育柱10-15分钟。
    8. 取出盖子和锁,并使洗涤缓冲液通过柱(参见图8),然后用4ml DEAE洗涤缓冲液pH 4洗涤(参见食谱)。密封如前所述,并在室温下在摇摆板上孵育10-15分钟。


      图8.允许洗涤缓冲区通过列

    9. 取出盖子和锁,并使洗涤缓冲液通过柱子,然后用3ml 0.1M NaCl(低亲和力)或0.25M NaCl(高亲和力)洗涤。如前所述密封,并在室温下在摇摆板上孵育15-30分钟。
    10. 去除盖子和锁,并允许洗涤缓冲液通过柱子。使DEAE树脂完全排出,每列加入5.4 ml 1.5 M NaCl。在室温下密封并在摇摆板上孵育1小时,以有效地洗脱GAG。
    11. 在10ml圆底管中收集洗脱液。通过SpeedVac离心将洗脱液的体积减少到约1.7ml。

      注意:


      1. 确保在应用新缓冲区之前,列底部已被密封

      2. 可以使用石蜡膜来密封色谱柱,但所提供的锁非常紧凑,易于处理。
      3. 重载DEAE列可以减少恢复。我们根据DEAE对高度硫酸化肝素的结合能力计算所需的床体积,以避免过载。但是,这些计算可能需要适用于其他样本。

  5. 脱盐样品
    使用NAP-10柱进行脱盐:
    1. 根据制造商的说明,清洗具有至少六列体积ddH 2 O的色谱柱。
    2. 确定浓缩洗脱液的体积并加载。
      注意:不能超过0.9毫升的样品一次可以脱盐,所以如果体积超过这一点,装载一半的洗脱液。
    3. 在样品进入色谱柱后添加ddH <2> O(步骤E2和E3的总体积应为1ml)。
    4. 将1.0-1.3ml ddH 2 O(1.4ml减去步骤E3中使用的体积)的Elute GAG放入10ml圆底管中。
    5. 洗涤具有至少七个柱体积的ddH 2 O的柱,以便制备它们用于下一个洗脱液并重复脱盐程序。
    6. 在SpeedVac离心机中完全干燥样品。

      注意:

      1. NAP-10色谱柱可以重复使用。用至少七列体积的ddH 将色谱柱洗涤,并在室温下将其储存在20%EtOH中。 />
      2. 为了避免样品之间的污染,也可以用1ml 5M NaCl洗涤柱,然后用ddH 2 /以除去任何剩余的高分子量污染物。
      3. 干燥的GAG通常具有“蓬松”的白色外观,而残留的盐是白色的,并且是结晶的。将疑似含有盐的样品溶解在100μlddH 2 中,并将1μl样品与0.1%AgNO / EM> <子> 3 。如果溶液中出现白云(类似于或强于含有1μl0.1 M NaCl的平行管),则将样品溶解在500μlddH 2 并重复脱盐。对所有并行样本执行此步骤,以确保以相同的方式对其进行处理。

  6. GAG降解和HS纯化
    我们使用的肝素酶也显示出弱CS降解活性,这就是为什么在HS消化和分析之前彻底去除CS是重要的:
    1. 在ddH 2 O中重构脱盐样品。
    2. 每管加入20μl5x软骨蛋白酶缓冲液(最终体积:100μl)(参见食谱)。
    3. 每mg干燥起始原料添加约1 mU软骨素酶(CSase)ABC。
    4. 在37℃孵育过夜(最少16小时)。
    5. 在96-100℃下热灭活CSase ABC 10分钟,并以最大速度离心样品10分钟。
    6. 如果不进行HS分析,将完整的样品转移到HPLC管中,并用ddH 2 O调节体积至100μl,以补偿孵育时间中的任何损失。进行“数据分析”中所述的二糖分析。
    7. 如果另外关于HS组成的信息将被确定,可以将5-20%的样品保存在用于CS组成分析的HPLC管中。
    8. 用ddH 2 O和5 M NaCl将剩余样品的体积调节至500μl,达到0.1 M NaCl的终浓度。
    9. 根据程序D所述准备DEAE柱(200μl床体积)
    10. 加载样品,密封柱,并在4℃下摇摆孵育1小时。
    11. 用1.2ml DEAE洗涤缓冲液pH8,1.2ml DEAE洗涤缓冲液pH4和1ml 0.1M NaCl洗涤柱子,无需进一步培养。
    12. 将洗涤缓冲液从柱中排出,并在室温下摇摆,将柱与1.8ml 1.5M NaCl孵育1小时,洗脱HS。
    13. 在2ml反应管中收集洗脱液,并将它们在SpeedVac离心机中浓缩至600-800μl
    14. 按照程序E所述,在NAP-10柱上脱盐。
      注意:样品中HS的含量远低于CS,因此干燥后几乎不会有沉淀。
    15. 将脱盐的样品重新置于100μlddH 2 O中,将其分成2x 50μl等分试样,并向其中加入49μl2x HS消化缓冲液。
    16. 加入1μl肝素酶混合物(每个肝素酶I,肝素钠酶II和肝素酶III为0.4mU)至一个等分试样和1μl1x HS消化缓冲液(参见Recipes)至另一份。
      注意:未消化的样品在HPLC运行中用作背景控制,参见“数据分析”)。
    17. 在37℃下孵育所有样品过夜(超过16小时)。
    18. 如步骤F5和F6所述,热灭活,离心并将样品转移到HPLC管中

      注意:

      1. HS和CS消化缓冲液可以等分并储存在-20°C以延长保质期。
      2. 其他缓冲液在室温下储存。
      3. CSase ABC(-20℃)和肝素裂解酶(-80℃)以5-20μl的等分试样储存,并避免多次冷冻。

数据分析

  1. 反相离子配对HPLC
    二染色体分离的条件(由CSase ABC消化产生或通过用肝素裂解酶I,II和III的混合物切割产生)和用2-氰基乙酰胺进行后柱衍生化的条件由Ledin等人( 2004),并由Dagalv等人(2011)轻微修改。
    1. 使用D-7000 HPLC系统管理器(HSM)软件4.1版,使用相同样品系列分析的二糖标准物,根据其洗脱位置和峰值大小来鉴定和定量峰。
      注意:准备不同类型的HS和CS二糖作为储备溶液的相似浓度(μg/μl),并混合以产生HS和CS标准品。为D-7000 HSM软件添加倍增因子,以解释二糖的不同分子质量。
    2. 在我们手中,HS样本中的背景噪声(CS样本中不存在)需要将非酶处理样品用作背景对照;从酶处理样品的色谱图中减去这一点。
    3. 将经过处理和未处理的样品直接相互连接起来,以最小化运行之间缓冲区条件的变化。
    4. D-7000 HSM软件将数据导出到Excel文件中。我们建议使用“宏”(Excel的编程指令集)根据相关标准的信号计算每峰值的总pmol。色谱图以及计算结果如图9所示

      图9.由D-7000 HSM软件导出的色谱图(上图)和计算(下图)

    5. 使用总pmol值计算每个样品中某种二糖(二糖组成)的相对量。
    6. 总硫酸化根据硫酸二糖的相对量计算,因此以每100个二糖的硫酸化度计算。
    7. 为了量化给定样品的总回收率,在校正注射样品的比例后,回收的GAG的量(以pmol计)可以归一化为mg干重。
      注意:一般来说,注射10-90μl以确保色谱柱不会过载。
    8. 作为干重的替代方案,样品也可以在Benzonase处理前对DNA含量进行归一化(参见方法C,步骤C7)。我们根据制造商的说明使用Quant-iT TM 宽范围测定试剂盒(从Molecular Probes购买),并对使用此方法的结果感到满意。
    9. 当比较野生型和突变株时,我们使用纯化和平行分析的样品。我们认为在不同日子生长和分离的样品是独立实验,通常进行至少三次独立实验
  2. 统计分析和数据呈现
    1. 所有图形都是使用GraphPad Prism版本5.0b为Mac OS X生成的(GraphPad Software,San Diego California USA, www.graphpad .com )。
    2. 图表显示平均值和误差条定义平均值(SEM)的标准误差。
    3. 在可能的情况下执行单尾Mann-Whitney测试,并且

      0.05被认为是重要的

笔记

  1. 生长条件将影响GAG组成,最有可能是由于C期间GAG结构的变化。线虫开发。因此,我们建议只比较在类似条件下生长的蠕虫。比较同一发育阶段的动物,大大提高了我们手中的再现性。
  2. 即使NAP-10色谱柱可以重复使用,也不应该保留高分子量分子,我们建议仅对相似的样品重复使用色谱柱,而不是为了避免污染物质。
  3. 当分析来自线虫的GAGs的链长时,我们注意到它们似乎比哺乳动物的短。因此,此处提出的方案可能需要优化从其他物种中分离硫酸化和非硫酸化GAG

食谱

注意:除非另有说明,否则缓冲液用非高压灭菌的去离子水制备并在室温下储存。灭菌的缓冲液经高压灭菌(120℃,20分钟)或通过0.45μm过滤器

  1. C的媒体。线虫生长和处理
    1. 1 M CaCl 2
      1.47g CaCl 2
      将体积调整为10 ml
    2. 5 N KOH
      14.028克KOH
      将体积调整为50 ml
    3. 1 M MgSO 4
      12.324g MgSO 4
      将体积调整为50 ml
    4. 胆固醇溶液
      在99.5%乙醇中5mg / ml 过滤灭菌
    5. 1M磷酸钾缓冲液pH 6.0(KPO 4
      120g KH 2 PO 4
      27.55克K 2/2 HPO 4 / 3H 2/1 / 1L
      用1M K 2 HPO 4
      调节pH至6.0 高压灭菌器
    6. 丰富的线虫生长培养基(RNGM)
      1.5克NaCl
      7.5 g琼脂糖
      3.75克Bacto蛋白胨每500毫升
      高压灭菌器
      冷却至不到55°C,然后加入:
      500μl胆固醇
      500μl1M MgSO 4
      500μl1M CaCl 2
      12.5ml 1M KPO 4缓冲液
      在分配到所需尺寸的培养皿之前仔细混合 干燥板在室温下储存在4°C
    7. LB琼脂
      2.5克细菌琼脂
      每250毫升5个LB片剂
      高压灭菌器
      储存于4°C
    8. 2x YT媒体
      1.6克Bacto蛋白胨
      1克细菌酵母提取物 每100ml 100ml 0.5g NaCl 高压灭菌器
      储存于4°C
    9. M9缓冲区
      5克NaCl
      15.12g Na 2 HPO 4 <7H 2 O O
      3g KH 2/3 PO 4/1/1/1 高压灭菌器
      加入1ml 1M MgSO 4 / / 4
  2. GAG净化缓冲液
    注意:用于洗涤和从DEAE柱洗脱的所有缓冲液通过0.45μm过滤器过滤
    1. 20%乙醇
      100 ml 99%乙醇 调节音量至500 ml
    2. 1 M MgCl 2
      2.03g MgCl 2
      将体积调整为10 ml
    3. 库存解决方案
      1. 10%Triton X-100
        5毫升Triton X-100(小心,非常黏稠!)
        将体积调整为50 ml
      2. 5 M NaCl
        292.2克NaCl
        将音量调整为1 L
        注意:过滤5 M NaCl是对所有DEAE缓冲区进行过滤的替代方法。
      3. 1 M CaCl 2(见配方A1)
      4. 1 M Tris-HCl
        121.14克Tris
        用6N HCl将pH调节至7.5 将音量调整为1 L
        高压灭菌器
    4. 蛋白酶缓冲液(50mM Tris / HCl pH 8.0,1mM CaCl 2,1%Triton X-100) 5 ml 1M Tris-HCl
      100μl1M CaCl 2
      在80ml ddH 2 O上的10ml 10%Triton X-100
      用6M HCl调节pH值
      将体积调整为100 ml
      储存于4°C
    5. DEAE洗脱缓冲液(1.5M NaCl)
    6. DEAE洗涤缓冲液(0.1M NaCl或0.25M NaCl)
    7. DEAE洗涤缓冲液,pH8(50M Tris / HCl pH 8.0,0.1M NaCl)
      25 ml 1 M Tris-HCl
      在400ml ddH 2 O上的10ml 5M NaCl
      用6M HCl调节pH值
      调节音量至500 ml
    8. DEAE洗涤缓冲液,pH 4(50mM NaOAc pH 4.0,0.1M NaCl) 25 ml 1 M NaOAc
      在400ml ddH 2 O上的10ml 5M NaCl
      用1M乙酸调节pH值
      调节音量至500 ml
    9. 0.1%AgNO 3
      100mg AgNO 3
      将体积调整为100 ml
    10. 5x软骨素酶ABC缓冲液(0.2M Tris-醋酸盐pH8.0)
      1.211g Tris在40ml ddH 2 O中的Tris- / / 用1M乙酸调节pH值
      将体积调整为50 ml
    11. 2x肝素酶裂解酶缓冲液(10mM HEPES pH 7.0,2mM CaCl 2)
      119.2mg HEPES
      在40ml ddH 2 O上的100μl1M CaCl 2
      用6M HCl调节pH值
      将体积调整为50 ml

致谢

来自瑞典研究委员会(至L.K.),德国学术交流处(至T.D.)和StiftelsenförProteoglykanforskning(至L.K.)的资助得到承认。 GAG纯化由Ledin等人描述,JBC 2004(doi:10.1074 / jbc.M405382200)和修饰用于秀丽隐杆线虫的方案首先由Dierker >等人,Sci。 Rep。2016(doi:10.1038 / srep34662)。

参考

  1. 主教,JR,Schuksz,M.和Esko,JD(2007)。&nbsp; 硫酸肝素蛋白聚糖微调哺乳动物生理学自然 446(7139):1030-1037。
  2. Bulow,HE和Hobert O.(2006)。糖胺聚糖的分子多样性塑造动物发育。 Annu Rev Cell Dev Biol 22:375-407。
  3. Dagalv,A.,Holmborn,K.,Kjellen,L.和Abrink,M。(2011)。硫酸乙酰肝素/肝素生物合成酶N-脱乙酰酶/ n-磺基转移酶1的降低表达导致肥大细胞肝素的硫酸化增加。 em> 286(52):44433-44440。
  4. Dierker,T.,Shao,C.,Haitina,T.,Zaia,J.,Hinas,A.,and Kjellen,L.(2016)。&nbsp; 线虫加入硫酸软骨素合成生物体家族:鉴定秀丽隐杆线虫中的活性软骨素磺基转移酶。 6:34662.
  5. Holberorn,K.,Habicher,J.,Kasza,Z.Eriksson,AS,Filipek-Gorniok,B.,Gopal,S.,Couchman,JR,Ahlberg,PE,Wiweger,M.,Spillmann,D.,Kreuger ,J.,and Ledin,J。(2012)。&lt; a class =“ke-insertfile”href =“https://www.ncbi.nlm.nih.gov/pubmed/22869369”target =“_ blank” >关于硫酸软骨素和硫酸乙酰肝素在斑马鱼咽软骨形态发生中的作用和调节。 J Biol Chem 287:33905-33916。
  6. Kiselova,N.,Dierker,T.,Spillmann,D.和Ramstrom,M。(2014)。一种用于分析硫酸化糖胺聚糖的基于质谱的自动化筛选方法。生物化学Biophys Res Commun 450 :598-603。
  7. Lawrence,R.,Olson,SK,Steele,RE,Wang,L.,Warrior,R.,Cummings,RD和Esko,JD(2008)。&nbsp; 通过定量聚糖还原同位素标记检测出的糖胺聚糖精细结构的进化差异 J. Biol Che m 283(48):33674-33684。
  8. Ledin,J.,Staatz,W.,Li,JP,Götte,M.,Selleck,S.,Kjellén,L。和Spillmann,D。(2004)。硫酸肝素结构在具有遗传修饰的硫酸乙酰肝素生产的小鼠中。 em> 279(41):42732-42741。
  9. Shao,C.,Shi,X.,Phillips,JJ和Zaia,J。(2013)。&nbsp; 来自组织学组织表面的糖胺聚糖的质谱谱图。分析化学 85(22):10984-10991。
  10. Townley,RA和Bulow,HE(2011)。&nbsp; 在秀丽隐杆线虫中的肝素修饰网络的遗传分析。 J Biol Chem 286:16824-16831 。
  11. Toyoda,H.,Kinoshita-Toyoda,A.,and Selleck,SB(2000)。&lt; a class =“ke-insertfile”href =“https://www.ncbi.nlm.nih.gov/pubmed/ 10644674“target =”_ blank“>果蝇中的糖胺聚糖和秀丽隐杆线虫的结构分析,并证明与果蝇相关的tout-velu,一种果蝇基因EXT肿瘤抑制因子,影响体内硫酸肝素。 。J.Bioll Chem 275(4):2269-2275。
  12. Yamada,S.,Sugahara,K.和Ozbek,S。(2011)。糖胺聚糖的进化。 Commun Integr Biol 4(2):150-158。
  13. Yamada,S.,Van Die,I.,Van den Eijnden,DH,Yokota,A.,Kitagawa,H.,and Sugahara,K。(1999)。&lt; a class =“ke-insertfile”href = https://www.ncbi.nlm.nih.gov/pubmed/10526159“target =”_ blank“>在秀丽隐杆线虫中的糖胺聚糖的演示 FEBS Lett 459(3):327-331。
  14. Zhang,L.(2010)。分子生物学和翻译学进展科学。 Elsevier 93:1-17。
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引用:Dierker, T. and Kjellén, L. (2017). Separation and Purification of Glycosaminoglycans (GAGs) from Caenorhabditis elegans. Bio-protocol 7(15): e2437. DOI: 10.21769/BioProtoc.2437.
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