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Oct 2017

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Liposome Flotation Assay for Studying Interactions Between Rubella Virus Particles and Lipid Membranes
用于风疹病毒颗粒和脂膜相互作用研究的脂质体浮选分析法   

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Abstract

Rubella virus (RuV) is an enveloped, positive-sense single-stranded RNA virus that is pathogenic to humans. RuV binds to the target cell via the viral envelope protein E1, but the specific receptor molecules on the target cell are yet to be fully elucidated. Here, we describe a protocol for liposome flotation assay to study direct interactions between RuV particles and lipid membranes in a qualitative manner. Interactions are examined by a Nycodenz density gradient fractionation using UV-inactivated RuV particles and fluorescent-labeled liposomes consisting of pure lipids. Fractionated RuV particles are detected using standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blot analysis for viral proteins. On the Nycodenz gradient, RuV particles bound to liposomes shift to lower density fractions than unbound RuV particles. Using this protocol, we provide compelling evidence that, at neutral pH in a calcium-dependent manner, RuV particles bind to lipid membranes containing both sphingomyelin (SM) and cholesterol in certain cell types.

Keywords: Liposome flotation assay (脂质体浮选分析法), Rubella virus (风疹病毒), Virus particles (病毒颗粒), Liposomes (脂质体), Lipids (脂类), Sphingomyelin (鞘磷脂), Cholesterol (胆固醇), Virus-Lipid interaction (病毒脂类相互作用)

Background

Rubella virus is the causative agent of ‘rubella’, an acute and relatively mild systemic infection and ‘congenital rubella syndrome’, a trans-placental fetal infection leading to serious birth defects (Hobman, 2013). Elucidation of molecular mechanisms of RuV entry is essential for understanding viral pathology and helpful for developing anti-RuV drugs. Though previous studies have suggested that membrane lipids of host cells serve as RuV receptors (Mastromarino et al., 1989 and 1990; DuBois et al., 2013), the detailed mechanism remains unknown. Recently, we found that RuV binds to erythrocytes and lymphoid cells in a calcium-dependent manner, and that the calcium-dependent viral binding is impaired after treatment of these cells with sphingomyelinase or cholesterol-adsorbent methyl-β-cyclodextrin, suggesting that SM and cholesterol of the host plasma membrane are critical for binding (Otsuki et al., 2018). To obtain compelling biochemical evidence, we established an assay system to detect interactions between RuV particles and lipids.

Representative biochemical assays widely applied for studying interactions between proteins and lipids are liposome co-sedimentation and co-flotation assays (Zhao and Lappalainen, 2012). Provided that RuV particles and liposomes form aggregates pelleted by low-speed centrifugation in analogy with viral hemagglutination, we initially tried to apply liposome co-sedimentation assay. Unfortunately, our trial of the co-sedimentation assay showed that only a small amount of RuV particles was pelleted at 15,000 x g in the presence of any liposomes. Nevertheless, RuV particles tended to be less pelleted in the presence of liposomes containing both SM and cholesterol, compared with those containing either or neither of the two lipids, providing us with direction for the study. Following this, we devised a flotation assay that can be performed on a small scale. For this, we employed a protocol originally applied for characterization of phosphoinositide binding of the S. cerevisiae Hsv2 (homologous with swollen vacuole phenotype 2) protein (Busse et al., 2013). After making several modifications in the original protocol to optimize for RuV analysis, we have established the protocol described below. By analysis with this protocol, we revealed that both SM and cholesterol are responsible for the calcium-dependent membrane binding of RuV particles.

Materials and Reagents

  1. Round-bottom glass tubes (size: 16 x 100 mm) (AGC Techno Glass, IWAKI, catalog number: TST-SCR16-100 ) with screw caps (AGC Techno Glass, IWAKI, catalog number: 9998CAP415-15 )
  2. Round-bottom glass tubes (size: 12 x 75 mm) (AGC Techno Glass, IWAKI, catalog number: 9831-1207 )
  3. Polypropylene centrifuge tubes:
    15 ml (AS ONE, VIOLAMO, catalog number: 1-3500-21 )
    50 ml (Corning, Centristar, catalog number: 430829 )
  4. Polypropylene microfuge tubes:
    1.5 ml (FUKAE KASEI, Watson, catalog number: 131-415C )
    1.5 ml (Safe-Lock tubes, Eppendorf, catalog number: 0030 120.086 , for heating SDS-PAGE samples)
    2.0 ml (FUKAE KASEI, Watson, catalog number: 132-620C )
  5. Polycarbonate ultracentrifuge tubes (Beckman Coulter, catalog number: 343778 )
  6. Pipette tips (Quality Scientific Plastics):
    1-200 μl (Thermo Fisher Scientific, catalog number: 110-96RSNEW )
    100-1,000 μl (Thermo Fisher Scientific, catalog number: 111-NXL-R100S )
  7. Serological pipets (Costar stripette):
    5 ml (Corning, catalog number: 4487 )
    10 ml (Corning, catalog number: 4488 )
  8. Immune-Blot polyvinylidene fluoride (PVDF) membrane (Bio-Rad Laboratories, catalog number: 1620177 )
  9. Chromatography papers (Whatman 3MM Chr, GE Healthcare, catalog number: 3030-672 )
  10. Polystyrene containers (180 x 90 x 45 mm) (AS ONE, catalog number: 1-4698-09 )
  11. Black 96-well strip plate (Black Combiplate 8, Labsystems, catalog number: 95029450 )
    Note: This product has been discontinued.
  12. Parafilm (Bemis, catalog number: PM996 )
  13. 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) (50 mg/ml in chloroform, Avanti Polar Lipids, catalog number: 850375C )
    Note: Store at -30 °C.
  14. SM from egg (Avanti Polar Lipids, catalog number: 860061P )
    Note: Store at -30 °C. 
  15. Cholesterol (Sigma-Aldrich, catalog number: C8667 )
    Note: Store at -30 °C.
  16. L-α-Phosphatidylethanolamine-N-(lissamine rhodamine B sulfonyl) (Ammonium Salt) (Rhod PE) (1 mg/ml in chloroform, Avanti Polar Lipids, catalog number: 810146C )
    Note: Transfer the solution to a round-bottom glass tube with a screw cap. Seal the cap with parafilm to avoid evaporation. Protect from light and store at -30 °C. Approximate molar concentration calculated with molecular weight of predominant species (1275.678 g/mol): 0.8 mM.
  17. Chloroform (Wako Pure Chemical Industries, catalog number: 038-02601 )
    Caution: Chloroform is volatile and hepatotoxic. Thus, when using chloroform and its mixtures, one MUST deal with them in a fume hood or with alternative equipment for chemical safety.
  18. Methanol (Sigma-Aldrich, catalog number: 19-2410-4 )
  19. Ethanol (Wako Pure Chemical Industries, catalog number: 057-00456 )
  20. Water (ultrapure water, e.g., Milli Q)
  21. UV-inactivated RuV particles for hemagglutination inhibition test (RuV antigens) (Denka Seiken, catalog number: 310071 )
    Note: Store at -30 °C. After reconstitution with 1 ml/vial of water, store at 4 °C and use within 3 days. 
  22. Calcium chloride dihydrate (CaCl2•2H2O) (NACALAI TESQUE, catalog number: 06731-05 )
  23. Tris-buffered saline (10x, pH 7.4) (TBS) (NACALAI TESQUE, catalog number: 35438-81 )
  24. Protease inhibitor cocktail for use with mammalian cell and tissue extracts (100x) (NACALAI TESQUE, catalog number: 25955-11 )
  25. 5-(N-2,3-dihydroxypropylacetamido)-2,4,6-triiodo-N,N’-bis(2,3-dihydroxypropyl) isophthalamide (Nycodenz) (Sigma-Aldrich, catalog number: D2158 )
  26. Sodium dodecyl sulfate (SDS) (NACALAI TESQUE, catalog number: 02873-75 )
  27. Glycerol (Wako Pure Chemical Industries, catalog number: 075-00616 )
  28. Dithiothreitol (DTT) (NACALAI TESQUE, catalog number: 14112-94 )
  29. Bromophenol blue (Wako Pure Chemical Industries, catalog number: 029-02912 )
  30. Tris(hydroxymethyl)aminomethane (Tris) (NACALAI TESQUE, catalog number: 35434-21 )
  31. Glycine (NACALAI TESQUE, catalog number: 17109-64 )
  32. Hydrochloric acid (HCl) (Wako Pure Chemical Industries, catalog number: 080-01066 )
  33. 30% (w/v)-Acrylamide/bis mixed solution (37.5:1) (NACALAI TESQUE, catalog number: 06144-05 )
  34. Ammonium peroxodisulfate (APS) (NACALAI TESQUE, catalog number: 02627-34 )
  35. N,N,N',N'-Tetramethylethylenediamine (TEMED) (NACALAI TESQUE, catalog number: 33401-72 )
  36. Prestained protein markers such as PINK prestained protein ladders (NIPPON Genetics, catalog number: MWP02 ) and ExcelBandTM 3-color regular range protein maker (SMOBIO Technology, catalog number: PM2500 )
  37. Skim milk (BD, Difco, catalog number: 232100 )
  38. Polyoxyethylene(20) sorbitan monolaurate (Tween 20) (Wako Pure Chemical Industries, catalog number: 167-11515 )
  39. Goat anti-RuV virion (strain HPV-77) polyclonal antibody (Acris Antibodies, catalog number: BP1061 )
    Note: Store at 4 °C. For long time storage, aliquot and store at -30 °C.
  40. Mouse monoclonal anti-goat/sheep IgG–peroxidase antibody (Sigma-Aldrich, catalog number: A9452 )
    Note: Store at 4 °C. For long-time storage, aliquot and store at -30 °C.
  41. Immobilon Western Chemiluminescent HRP Substrate (Merck, catalog number: WBKLS0500 )
  42. Chloroform/methanol (19/1, v/v)
  43. 0.2 M CaCl2
  44. 1 M DTT
  45. 1% (w/v) bromophenol blue in 50% (v/v) ethanol
  46. 10% (w/v) SDS
  47. 1 M Tris-HCl pH 8.8
  48. 1 M Tris-HCl pH 6.7
  49. 10% (w/v) APS
  50. 20% (w/v) Tween 20
    Note: Store at 4 °C. Check bacterial or fungal contamination before use.
  51. Lipid stock solutions
  52. TBS (see Recipe 4)
  53. 2x TBS (see Recipe 5)
  54. 80% (w/v) Nycodenz/TBS (see Recipe 6)
  55. 30% (w/v) Nycodenz/TBS (see Recipe 7)
  56. 3x SDS sample buffer (see Recipe 8)
    Note: Aliquot and store at -30 °C. 
  57. 10% (w/v) polyacrylamide separating gel solution (see Recipe 9)
  58. 5% (w/v) polyacrylamide stacking gel solution (see Recipe 10)
  59. 10x Tris/Glycine (see Recipe 11)
  60. Electrode buffer (see Recipe 12)
  61. Transfer buffer (see Recipe 13)
  62. TBS-T (see Recipe 14)
  63. 5% (w/v) skim milk/TBS-T (see Recipe 15)
  64. 2% (w/v) skim milk/TBS-T (see Recipe 16)

Equipment

  1. Micropipettes durable against organic solvent dispensing (NICHIRYO, model: Nichipet EX Plus II, catalog numbers: 00-NPLO2-20 , 00-NPLO2-200 , 00-NPLO2-1000
  2. Pipet-Aid XP Pipette Controller (Drummond Scientific, catalog number: 4-040-101-J )
  3. Vortex mixer (Delta mixer, TAITEC, model: Se-08 )
  4. Nitrogen evaporator with water bath (Nakajima seisakusho, Co., Ltd., custom-made)
  5. Fume hood equipped with activated charcoal filters (Dalton, model: DC-183-100
  6. Probe-type ultrasonic processor (Hielscher Ultrasonics, model: UP50H )
  7. Micro refrigerated centrifuge (e.g., KUBOTA, model: 3520 )
  8. Electronic balance (Shimadzu, LIBROR, model: EB-340HW ; Mettler-Toledo International, model: ML802/52 )
  9. Ultracentrifuge (Beckman Coulter, model: OptimaTM TLX with TLS-55 rotor)
  10. Block heater for microfuge tubes (Nippon Genetics, Fast GeneTM, model: FG-02N )
  11. pH meter (TOA Electronics, model: HM-30S
  12. Microwave oven (TOSHIBA, model: ER-225 )
  13. Protein electrophoresis apparatus for SDS-PAGE (e.g., BIO CRAFT, model: BE-S28 for wide mini gels)
  14. Western blot apparatus (e.g., Bio-Rad Laboratories, CriterionTM blotter for wide mini gels, catalog number: 1704070 )
  15. Power supply (ATTO, model: AE-8450 , for SDS-PAGE; Bio-Rad Laboratories, model: 250/2.5 , for Western blotting)
  16. Tube rotator (SCINICS, model: RVM-101 )
  17. Reciprocal (TAITEC, model: Personal-11 ) and/or seesaw (TAITEC, model: Wave-SI ) shakers
  18. Chemiluminescent imaging system (ATTO, model: WSE-6200H LuminoGraph II)
  19. Microplate reader (BMG LABTECH, model: FLUOstar Optima )

Software

  1. ImageSaver 6 for Windows (ATTO Co.) as image acquisition software for WSE-6200H LuminoGraph II
  2. CS Analyzer 4 for Windows (ATTO Co.) as image analysis software
  3. Microsoft Excel 2016 (Microsoft Corp.) as spreadsheet software
  4. GraphPad Prism 7 (Graph Pad Software) as graph drawing software

Procedure

  1. Preparation of liposomes
    Conduct all manipulations at room temperature unless otherwise stated. When using samples containing fluorescent-labeled lipids, protect them from direct light whenever possible throughout the procedure.
    1. For preparation of 2x concentrated liposomes consisting of DOPC/SM/cholesterol/Rhod PE in a molar ratio of 8:2:3:0.05, prepare a stock solution of each lipid in organic solvent and then mix an appropriate volume of each stock solution in a round-bottom glass tube to obtain the final amounts: 1.6 μmol DOPC, 0.4 μmol SM, 0.6 μmol cholesterol, and 10 nmol Rhod PE (Figure 1A). For liposomes of other compositions, alter the amounts of lipid put into the glass tube appropriately (Table 1).

      Table 1. Lipid compositions of 2x concentrated liposomes. Each liposome contains an equal amount of phospholipids. DOPC is used as the matrix phospholipid because phosphatidylcholine is the most abundant type of phospholipid in the plasma membrane. The molar ratio of DOPC/SM/cholesterol of liposome A (8/2/3) is the same as that of the liposomes used in the previous study on sphingomyelin-specific toxin lysenin (Ishitsuka and Kobayashi, 2007). Liposome A and A’ are identical in composition. 

      adissolved in chloroform/methanol (19/1, v/v); bdissolved in chloroform; cadded at 1 ml per glass tube after evaporation.

    2. Evaporate organic solvent completely under nitrogen gas at a bath temperature of 35-40 °C using nitrogen evaporator set up in a fume hood. After at least 20 min of evaporation, lipid film is formed at the bottom of the glass tube (Figure 1B).
    3. Rehydrate the resultant lipid film with 1 ml of TBS-Ca (Figure 1C). To prepare liposomes without calcium ions, use TBS instead of TBS-Ca.


      Figure 1. Preparation of liposomes. A. Mixture of lipid solution before evaporation; B. Lipid film formed after evaporation; C. Lipid film rehydrated with TBS-Ca; D. Lipid suspension after sonication.

    4. Sonicate the mixture for 20-30 min with a probe-type sonicator with cooling in tap water (without ice). For UP50H ultrasonic processor, set an amplitude at 80% and a duty cycle at 50%.
      Note: The mixture looks turbid at the onset. After 30 min (for liposome A) or 20 min (for liposomes with other compositions) of sonication, the mixture becomes transparent. Vortex the tube every 10 min of sonication to restore liquid spattered on the inner wall of the tube.
    5. Transfer the lipid suspension to a 1.5-ml microfuge tube.
    6. Centrifuge the lipid suspension at 15,000 x g for 10 min at 25 °C with a micro refrigerated centrifuge to precipitate the lipid aggregates and debris from sonicator tip.
    7. Transfer the supernatant to a new 1.5 ml microfuge tube and centrifuge again under the same conditions. Repeat centrifugation until no visible pellet is formed.
    8. Transfer the supernatant to a new 1.5 ml microfuge tube and store the fraction as 2x concentrated liposome fraction at 4 °C. Protect from light and use within 3 days.

  2. Nycodenz density gradient centrifugation
    Interaction between RuV antigens and liposomes is assessed using Nycodenz density gradient fractionation. Schematic outline of this experiment is shown in Figure 2. Conduct all manipulations at room temperature unless otherwise stated. Warm all the reagents to room temperature before use. To omit calcium ions from each reaction, use TBS-based reagents instead of TBS-Ca-based ones.


    Figure 2. Schematic outline of Nycodenz density gradient fractionation

    1. Mix the following components in a polycarbonate ultracentrifuge tube:
      42 μl of Water
      75 μl of 2x TBS-Ca
      3 μl of 100x protease inhibitor cocktail
      30 μl of RuV antigens
      (Total volume: 150 μl)
      Notes: 
      1. RuV antigens must be added at last. Mix well by pipetting before and after the addition of RuV antigens. 
      2. RuV antigens contain bovine serum albumin added as an additive by the manufacturer. Its concentration is not disclosed.
    2. Add 150 μl of 2x concentrated liposome fraction prepared in Step A8 to the mixture, and mix well by pipetting.
    3. Incubate the mixture for 1 h, protecting from light.
    4. Mix the reaction mixture (300 μl) with 300 μl of 80% (w/v) Nycodenz/TBS-Ca by pipetting to obtain homogeneous solution. The resultant solution contains 40% (w/v) Nycodenz.
    5. Overlay the 40% (w/v) Nycodenz solution with 300 μl of 30% (w/v) Nycodenz/TBS-Ca, being careful not to mix them up.
    6. Overlay the 30% (w/v) Nycodenz solution with 300 μl of TBS-Ca, being careful not to mix them up.
    7. Weigh each tube and swinging bucket. Balance weight of two sets of tubes and buckets that will be placed diagonally in TLS-55 rotor in 0.01 g scale by adding TBS-Ca to a tube.
      Note: Although the weights of the buckets are initially matched by the manufacturer, the buckets need to be balanced in weight because they can become worn over time.
    8. Set each tube carefully into a swinging bucket.
    9. Hang the bucket into a TLS-55 rotor carefully, then set the rotor in an Optima TLX ultracentrifuge.
    10. Centrifuge the samples at 55,000 rpm (259,000 x gmax) for 5 h at 25 °C.
    11. Remove each tube carefully from the buckets.
    12. Collect 50 μl aliquots from the top of the gradient using P-200 micropipette with a 1-200 μl tip. Always set the point of the pipette tip just below the aqueous surface (≤ 1 mm) and draw the solution slowly in a circular motion, being careful not to suction air. 
    13. Combine two 50 μl aliquots in a 1.5 ml microfuge tube (i.e., ~100 μl/tube). Although 100 μl aliquots can be taken at once with a micropipette, the authors recommend 50 μl aliquots: A short pipetting stroke is easier to control, thereby lowering the risk of disturbing the gradient. Fractionate each sample into 12 fractions. When the volume of the last fraction is smaller than 100 μl, adjust the volume to ~100 μl with TBS-Ca.
      Note: Mark ~100 μl line on 1.5-ml tubes for the last fractions by using 100 μl of water in advance.
    14. Transfer 30 μl of each fraction into a 1.5-ml microfuge tube, then mix with 15 μl of 3x SDS sample buffer. Store at -30 °C until SDS-PAGE analysis. 15 μl of the resultant solution contains 10% (10 μl) of each collected fraction.
    15. Dilute 24 μl of RuV antigens with 56 μl of TBS-Ca, then mix with 40 μl of 3x SDS sample buffer. Store at -30 °C until SDS-PAGE analysis. 15 μl of the resultant solution contains 3 μl of RuV antigens, which corresponds to 10% of those input into each ultracentrifuge tube. Store the residual fractions (~70 μl/tube) at 4 °C until measurement of fluorescence.

  3. SDS-PAGE and Western blot analysis
    Distribution of RuV particles among the collected fractions is analyzed using standard SDS-PAGE followed by Western blot analysis for RuV proteins. Conduct all manipulations at room temperature unless otherwise stated.
    1. See a basic protocol for SDS-PAGE (He, 2011). Prepare a 10% (w/v) polyacrylamide separating gel using a gel casting device of a protein electrophoresis apparatus according to the manufacturer’s instructions. 
    2. Prepare a 5% (w/v) polyacrylamide stacking gel, similarly.
    3. Assemble the gel into an electrophoresis device and pour electrode buffer.
    4. Heat samples for SDS-PAGE analysis prepared in Steps B14 and B15 at 95 °C for 5 min using a block heater, then cool to room temperature. Spin at 15,000 x g for ~10 sec at 25 °C.
    5. Load 15 μl of the resultant samples per lane of the gel. Load 5 μl of prestained molecular markers similarly.
    6. Perform electrophoresis at 100-200 V constant according to the manufacturer’s instruction.
    7. After electrophoresis, place the gel into a clean container and shake it in transfer buffer for 10 min using reciprocal or seesaw shakers. Pre-wet a PVDF membrane with methanol and shake it similarly.
    8. Set up a sandwich of layers, including chromatography papers, the gel, and the membrane, soaking in transfer buffer.
    9. Place the sandwich in a chamber of a Western blot apparatus filled with transfer buffer.
    10. Transfer proteins from the gel to the membrane electrophoretically at 100 V constant for 30 min in a cold room at 4 °C according to the manufacturer’s instructions.
    11. Place the membrane in a clean container and soak in 5% (w/v) skim milk/TBS-T for 1 h with shaking.
    12. Wash the membranes three times for 5 min each in TBS-T.
    13. Place the membrane in a clean container and add the primary antibody (goat anti-RuV polyclonal antibody) diluted 1:4,000 with 2% (w/v) skim milk/TBS-T.
    14. Incubate the membrane at 4 °C overnight with shaking.
    15. Wash the membranes three times for 5 min each in TBS-T.
    16. Place the membrane in a clean container and add the secondary antibody (HRP-conjugated mouse anti-goat/sheep IgG) diluted1:4,000 with 2% (w/v) skim milk/TBS-T.
    17. Incubate the membrane for 1 h with shaking.
    18. Wash the membranes three times for 10 min each in TBS-T.
    19. Develop signal by incubating the membrane with Immobilon Western Chemiluminescent HRP Substrate or an equivalent reagent according to the manufacturer’s instructions.
    20. Detect the signal using a WSE-6200H LuminoGraph II image analyzer according to the manufacturer’s instructions.

  4. Measurement of fluorescence
    Distribution of liposomes among the collected fractions is analyzed based on fluorescence intensity of Rho PE. Warm the collected fractions and 2x concentrated liposome fractions to room temperature before measurement.
    1. Transfer 50 μl of each collected fraction (which corresponds to 50% of each fraction) and 2x concentrated liposome fraction (which corresponds to 33% of total input) into a well of a 96-well black microplate.
    2. Measure fluorescence intensity of Rhod PE in each well using a FLUOstar Optima microplate reader at 544 nm excitation and 590 nm emission in endpoint mode without shaking function according to the manufacturer’s instruction. 
    3. Save results in an excel file format.

Data analysis

  1. Visualize RuV proteins in the collected fraction using image acquisition and analysis software. The apparent molecular weights of RuV E1 (one of two envelope glycoproteins) and capsid proteins are 58-65 kDa and 33-38 kDa, respectively. Figure 3 shows the distribution of E1 and capsid proteins on Nycodenz density gradient fractionation in the presence or absence of various liposomes. RuV particles appear to be present in fractions that contained both proteins. The dense fractions (fractions 10-12) contained only the capsid protein, suggesting that RuV particles were absent from these fractions. Although RuV particles were mainly detected in fraction 6 in the absence of liposomes (Figure 3F), they shifted to fraction 4 in the presence of liposomes consisting of DOPC/SM/cholesterol (Figures 3A and 3A'). This shift toward lower densities was not observed when calcium ions were omitted from the reaction (Figure 3E), indicating that RuV particles interact with lipid membranes in a calcium-dependent manner, consistent with a previous study (Dubé et al., 2014). Furthermore, the shift was no longer observed when either SM or cholesterol was omitted from the liposomes (Figures 3B and 3C) and liposomes consisting of DOPC alone were used (Figure 3D), indicating that the calcium-dependent interaction between RuV particles and lipid membranes requires both SM and cholesterol in the membranes. In Figure 3, results of a separate experiment from that described in the original article (Otsuki et al., 2018) are shown. Similar results were obtained from three independent experiments.


    Figure 3. Distribution of RuV proteins on the Nycodenz gradient. RuV antigens (UV-inactivated RuV particles) were incubated with or without various compositions of liposomes in the presence (+) or absence (-) of calcium ions and then subjected to liposome flotation assays on the Nycodenz gradient. Viral E1 and capsid (C) proteins in each fraction were detected by Western blot analysis. An alphabetic code of each panel corresponds to that of liposomes described in Table 1 except for panel F, which indicates the result without liposomes. The values indicated are the amounts (nmol) of DOPC, SM, and cholesterol (Chol.) in each liposome fraction added per μl of RuV antigens. All of the liposomes also contained 0.05 nmol (per μl of RuV antigens) of Rhod PE. Asterisks indicate bovine serum albumin, which was an additive to RuV antigens. Two separate sets of centrifugation experiments are shown: One set, the upper four panels (A-D), and another set, the lower three panels (A'-F). Image acquisition was carried out under the same conditions (10 min of exposure in high sensitivity mode), and contrast of images was adjusted to the same extent (setting upper value at 40,000 and lower value at zero). Panels A and A' represent results from separate experiments using liposomes with the same lipid composition and the same experimental procedures. In this assay, the distribution of viral proteins was similar, but their overall signal intensity varied among the experiments. Though the overall signal intensity was not the main issue of this assay, a possible cause of this variability was an inconstant transfer efficiency in each gel.

  2. Quantify capsid protein using image analysis software and determine recovery of the protein in each fraction. For this purpose, E1 protein is not suitable, because a large amount of bovine serum albumin added as a preservative to RuV antigens overlaps substantially with E1 protein on a Western blot of the input fraction. Total recovery of capsid protein varied from ~10% to ~70% with experiments probably due to the semi-quantitative nature of Western blot analysis and/or degradation of the protein during or after ultracentrifugation. We did not find any correlation between the type of liposome and the recovery of capsid protein.
  3. To check distribution of liposomes, calculate % of Rhod PE recovered in each fraction using the data of fluorescent intensity. Figure 4 shows the distribution of liposomes among density gradient fractions. The majority of liposomes (~70% or more) were recovered in the top and second fractions irrespective of lipid compositions. Since floating liposomes can not be completely recovered, a small amount of them (< 10%) remained on the aqueous surface throughout the fractionation procedure and was recovered in the last fraction. In Figure 4, results of a separate experiment from that described in the original article (Otsuki et al., 2018) are shown. Similar results were obtained from three independent experiments.


    Figure 4. Distribution of liposomes on the Nycodenz gradient. Distribution of liposomes was monitored using the fluorescence intensity of Rhod PE. Values are expressed as a percentage (%) of the total fluorescence intensity of Rhod PE retrieved from each fraction of the gradient. Alphabetic codes correspond to liposomes described in Table 1.

Notes

  1. Homepage of Avanti Polar Lipids, Inc. (https://avantilipids.com/) is informative for beginners in handling of lipids.
  2. Homepage of Axis-Shield Density Gradient Media, a brand of Alere Technologies AS (http://www.optiprep.com/) provides general information about Nycodenz.
  3. Peak positions of RuV particles are mostly reproducible between experiments, but can vary depending on slight differences in the respective experimental conditions. Thus, samples to be directly compared should be prepared, centrifuged, and fractionated in parallel.

Recipes

Note: The water used in the following recipes is ultrapure water unless otherwise stated.

  1. DOPC stock solution (5 mg/ml)
    1. Put 1 ml of DOPC (50 mg/ml in chloroform) into a round-bottom glass tube with a screw cap
    2. Dilute with 9 ml of chloroform/methanol (19/1, v/v)
    3. Seal the cap with parafilm to lessen evaporation of the solvent
    4. Store at -30 °C and warm to room temperature before use
    Note: To obtain molar concentration of DOPC and SM (see below) stock solutions, determine the concentration of phosphorus in the solution according to Rouser et al. (1966).
  2. SM stock solution (5 mg/ml)
    1. Weigh 50 mg of SM (powder) and put into a round-bottom glass tube with a screw cap
    2. Dissolve SM in 10 ml of chloroform/methanol (19/1, v/v)
    3. Seal the cap with parafilm to lessen evaporation of the solvent
    4. Store at -30 °C. Warm to room temperature before use
  3. Cholesterol stock solution (2 mg/ml, calculated molar concentration: 5.17 mM)
    1. Weigh 20 mg of cholesterol (powder) and put into a round-bottom glass tube with a screw cap
    2. Dissolve cholesterol in 10 ml of chloroform/methanol (19/1, v/v)
    3. Seal the cap with parafilm to lessen evaporation of the solvent
    4. Store at -30 °C. Warm to room temperature before use
  4. TBS
    25 mM Tris-HCl pH 7.4
    137 mM NaCl
    2.68 mM KCl
    1. Add 2.5 ml of 10x TBS into a 50-ml centrifuge tube
    2. Fill up to 25 ml with water and mix well
    To prepare TBS containing 1 mM CaCl2 (TBS-Ca), add 125 μl of 0.2 M CaCl2 before filling up with water
    Store at 4 °C
  5. 2x TBS
    Dilute 400 μl of 10x TBS with 1.6 ml of water in a 2-ml microfuge tube
    To prepare 2x TBS-Ca, add 20 μl of 0.2 M CaCl2 (final concentration: 2 mM) and mix well
    Store at 4 °C
  6. 80% (w/v) Nycodenz/TBS
    1. Dissolve 8 g of Nycodenz with hot water in a 15-ml centrifuge tube
      Note: It requires time to dissolve such an amount of Nycodenz in less than 10 ml of water. Use hot water preheated by a microwave and mix intensively. Warm the tube occasionally with hot tap water. If hard-to-melt clumps appear, disrupt them by sonication in a bath-type sonicator.
    2. Add 1 ml of 10x TBS and fill up to 10 ml with water. Mix well and store at 4 °C.
    3. Before use, transfer an appropriate amount of the solution into a 2.0-ml microfuge tube and add 1/100 volume of 100x protease inhibitor cocktail. Mix well.
    To prepare 80% (w/v) Nycodenz/TBS-Ca, also add 1/200 volume of 0.2 M CaCl2 (final concentration: 1 mM) and mix well
  7. 30% (w/v) Nycodenz/TBS
    1. Dissolve 3 g of Nycodenz in hot water in a 15-ml centrifuge tube
    2. Add 1 ml of 10x TBS
    3. Fill up to 10 ml with water and mix well. Store at 4 °C
    4. Before use, transfer an appropriate amount of the solution into a 2.0-ml microfuge tube and add 1/100 volume of 100x protease inhibitor cocktail. Mix well
    To prepare 30% (w/v) Nycodenz/TBS-Ca, also add 1/200 volume of 0.2 M CaCl2 (final concentration: 1 mM) and mix well
  8. 3x SDS sample buffer
    1. Mix the following components in a 15-ml centrifuge tube:
      4.5 ml of Glycerol (final concentration: 30% [v/v])
      2.25 ml of 1 M DTT (final concentration: 150 mM)
      1.5 ml of 1% (w/v) bromophenol blue in 50% (v/v) ethanol (final concentration: 0.1% [w/v])
      2.81 ml of 1 M Tris-HCl pH 6.7 (final concentration: 187.5 mM)
    2. Dissolve 0.9 g of SDS (final concentration: 6% [w/v]) using a tube rotator
    3. Fill up to 15 ml with water and mix well
    4. Store at -30 °C and warm at 37 °C to dissolve SDS before use
  9. 10% (w/v) polyacrylamide separating gel solution
    1. Mix the following components in a 50-ml centrifuge tube:
      8.28 ml of Water
      11.25 ml of 1 M Tris-HCl pH 8.8 (final concentration: 375 mM)
      10 ml of 30% (w/v)-Acrylamide/bis mixed solution
      300 μl of 10% (w/v) SDS (final concentration: 0.1% [w/v])
      300 μl of 10% (w/v) APS (final concentration: 0.1% [w/v])
    2. Add 15 μ of TEMED (final concentration: 0.05% [v/v]) and mix well
    3. Pour into gel casting apparatus immediately
    Note: This volume is enough for two wide mini gels.
  10. 5% (w/v) polyacrylamide stacking gel solution
    1. Mix the following components in a 15-ml centrifuge tube:
      6.92 ml of Water
      1.25 ml of 1 M Tris-HCl pH 6.7 (final concentration: 125 mM)
      1.67 ml of 30% (w/v)-Acrylamide/bis mixed solution
      100 μl of 10% (w/v) SDS (final concentration: 0.1% [w/v])
      100 μl of 10% (w/v) APS (final concentration: 0.1% [w/v])
    2. Add 10 μ of TEMED (final concentration: 0.1% [v/v]) and mix well
    3. Cast immediately on separating gels
    Note: This volume is enough for two wide mini gels.
  11. 10x Tris/Glycine
    1. Dissolve 121 g of Tris and 576 g of glycine in about 3 L of water
    2. Fill up to 4 L with water and mix well
    3. Store at room temperature
  12. Electrode buffer
    1. Dilute 100 ml of 10x Tris/Glycine with about 800 ml of water
    2. Add 10 ml of 10% (w/v) SDS
    3. Fill up to 1 L with water and mix well
    4. Store at room temperature
  13. Transfer buffer
    1. Dilute 200 ml of 10x Tris/Glycine with 1.4 L of water
    2. Add 400 ml of methanol and mix well
    3. Keep on ice or at 4 °C
    Note: Do not add methanol to 10x Tris/Glycine before adding water. The mixture becomes cloudy.
  14. TBS-T
    1. Dilute 100 ml of 10x TBS with less than 900 ml of water
    2. Add 5 ml of 20% (w/v) Tween 20 to give a final concentration of 0.1% (w/v)
    3. Fill up to 1 L with water and mix well
    4. Store at 4 °C and warm to room temperature before use
  15. 5% (w/v) skim milk/TBS-T
    Dissolve 2.5 g of skim milk in 50 ml of TBS-T in a 50 ml centrifuge tube using a tube rotator
    Note: Mix the tube for at least 1 h for complete dissolving. Prepare on the day of use and store at 4 °C. Use within 2 days.
  16. 2% (w/v) skim milk/TBS-T
    Dissolve 1 g of skim milk in 50 ml of TBS-T in a 50 ml centrifuge tube using a tube rotator
    Note: Mix the tube for at least 1 h for complete dissolving. Prepare on the day of use and store at 4 °C. Use within 2 days.

Acknowledgments

This protocol has been used in Otsuki et al. (2018). This study was supported by a grant to KH from AMED-CREST (Japan Agency for Medical Research and Development, Core Research for Evolutional Science and Technology, grant number: JP18gm0910005). This protocol was adapted from the work by Busse et al. (2013).

Competing interests

The authors declare that they have no conflict of interest.

References

  1. Busse, R. A., Scacioc, A., Hernandez, J. M., Krick, R., Stephan, M., Janshoff, A., Thumm, M. and Kuhnel, K. (2013). Qualitative and quantitative characterization of protein-phosphoinositide interactions with liposome-based methods. Autophagy 9(5): 770-777. 
  2. Dubé, M., Rey, F. A. and Kielian, M. (2014). Rubella virus: first calcium-requiring viral fusion protein. PLoS Pathog 10(12): e1004530. 
  3. DuBois, R. M., Vaney, M. C., Tortorici, M. A., Kurdi, R. A., Barba-Spaeth, G., Krey, T. and Rey, F. A. (2013). Functional and evolutionary insight from the crystal structure of rubella virus protein E1. Nature 493(7433): 552-556.
  4. He, F. (2011). Laemmli-SDS-PAGE. Bio-protocol Bio101: e80. 
  5. Hobman, T. C. (2013). Rubella virus. In: Knipe, D. M., Howley, P. M., Cohen, J. I., Griffin, D. E., Lamb, R. A., Martin, M. A., Racaniello, V. R., Roizman, B. (Eds.). Fields virology. 6th edition. vol 1. p 687-711. Lippincott Williams & Wilkins, Philadelphia, PA.
  6. Ishitsuka, R. and Kobayashi, T. (2007). Cholesterol and lipid/protein ratio control the oligomerization of a sphingomyelin-specific toxin, lysenin. Biochemistry 46(6): 1495-1502. 
  7. Mastromarino, P., Cioè, L., Rieti, S. and Orsi, N. (1990). Role of membrane phospholipids and glycolipids in the Vero cell surface receptor for rubella virus. Med Microbiol Immunol 179(2): 105-114. 
  8. Mastromarino, P., Rieti, S., Cioè, L. and Orsi, N. (1989). Binding sites for rubella virus on erythrocyte membrane. Arch Virol 107(1-2): 15-26. 
  9. Otsuki, N., Sakata, M., Saito, K., Okamoto, K., Mori, Y., Hanada, K. and Takeda, M. (2018). Both sphingomyelin and cholesterol in the host cell membrane are essential for Rubella virus entry. J Virol. 92(1): e01130-17.
  10. Rouser, G., Siakotos, A. N. and Fleischer, S. (1966). Quantitative analysis of phospholipids by thin-layer chromatography and phosphorus analysis of spots. Lipids 1(1): 85-86. 
  11. Zhao, H. and Lappalainen, P. (2012). A simple guide to biochemical approaches for analyzing protein-lipid interactions. Mol Biol Cell 23(15): 2823-2830.

简介

风疹病毒(RuV)是一种包膜的正义单链RNA病毒,对人类具有致病性。 RuV通过病毒包膜蛋白E1与靶细胞结合,但靶细胞上的特异性受体分子尚未完全阐明。在这里,我们描述了脂质体浮选测定的方案,以定性方式研究RuV颗粒和脂质膜之间的直接相互作用。使用UV-灭活的RuV颗粒和由纯脂质组成的荧光标记的脂质体通过Nycodenz密度梯度分级检查相互作用。使用标准十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳(SDS-PAGE)检测分级的RuV颗粒,然后对病毒蛋白进行Western印迹分析。在Nycodenz梯度上,与未结合的RuV颗粒相比,与脂质体结合的RuV颗粒转移至较低密度的部分。使用该方案,我们提供了令人信服的证据,即在中性pH下以钙依赖性方式,RuV颗粒与某些细胞类型中含有鞘磷脂(SM)和胆固醇的脂质膜结合。

【背景】 风疹病毒是“风疹”的致病因子,“风疹”是一种急性且相对轻微的全身性感染和“先天性风疹综合征”,一种导致严重出生缺陷的转胎胎儿感染(Hobman,2013)。阐明RuV进入的分子机制对于了解病毒病理学和帮助开发抗RuV药物是必不可少的。虽然以前的研究表明宿主细胞的膜脂质作为RuV受体(Mastromarino et al。,1989和1990; DuBois et al。,2013),但详细的机制仍然未知。最近,我们发现RuV以钙依赖性方式与红细胞和淋巴细胞结合,并且在用鞘磷脂酶或胆固醇吸附剂甲基-β-环糊精处理这些细胞后钙依赖性病毒结合受损,表明SM和宿主质膜的胆固醇对于结合是关键的(Otsuki 等人,,2018)。为了获得令人信服的生化证据,我们建立了一种检测系统来检测RuV颗粒与脂质之间的相互作用。

广泛应用于研究蛋白质和脂质之间相互作用的代表性生物化学分析是脂质体共沉淀和共浮选分析(Zhao和Lappalainen,2012)。如果RuV颗粒和脂质体形成通过类似于病毒血细胞凝集的低速离心沉淀的聚集体,我们最初尝试应用脂质体共沉降测定法。不幸的是,我们的共沉淀测定试验显示,在任何脂质体存在下,只有少量RuV颗粒在15,000 x g 沉淀。然而,与含有两种脂质或不含两种脂质的脂质体相比,RuV颗粒在含有SM和胆固醇的脂质体存在下往往更少沉淀,这为我们提供了研究方向。在此之后,我们设计了一种可以小规模进行的浮选分析。为此,我们采用了最初用于表征 S的磷酸肌醇结合的方案。 cerevisiae Hsv2(与肿胀的液泡表型2同源)蛋白质(Busse et al。,2013)。在对原始方案进行若干修改以优化RuV分析后,我们建立了下述方案。通过该方案的分析,我们发现SM和胆固醇都是RuV颗粒的钙依赖性膜结合的原因。

关键字:脂质体浮选分析法, 风疹病毒, 病毒颗粒, 脂质体, 脂类, 鞘磷脂, 胆固醇, 病毒脂类相互作用

材料和试剂

  1. 圆底玻璃管(尺寸:16 x 100 mm)(AGC Techno Glass,IWAKI,目录号:TST-SCR16-100),带螺旋盖(AGC Techno Glass,IWAKI,目录号:9998CAP415-15)
  2. 圆底玻璃管(尺寸:12 x 75 mm)(AGC Techno Glass,IWAKI,目录号:9831-1207)
  3. 聚丙烯离心管:
    15毫升(AS ONE,VIOLAMO,目录号:1-3500-21)
    50毫升(Corning,Centristar,目录号:430829)
  4. 聚丙烯微量离心管:
    1.5毫升(FUKAE KASEI,Watson,目录号:131-415C)
    1.5 ml(Safe-Lock试管,Eppendorf,目录号:0030 120.086,用于加热SDS-PAGE样品)
    2.0毫升(FUKAE KASEI,Watson,目录号:132-620C)
  5. 聚碳酸酯超速离心管(Beckman Coulter,目录号:343778)
  6. 移液器吸头(Quality Scientific Plastics):
    1-200μl(Thermo Fisher Scientific,目录号:110-96RSNEW)
    100-1,000μl(Thermo Fisher Scientific,目录号:111-NXL-R100S)
  7. 血清移液管(Costar stripette):
    5毫升(康宁,目录号:4487)
    10毫升(康宁,目录号:4488)
  8. Immunune-Blot聚偏二氟乙烯(PVDF)膜(Bio-Rad Laboratories,目录号:1620177)
  9. 色谱纸(Whatman 3MM Chr,GE Healthcare,目录号:3030-672)
  10. 聚苯乙烯容器(180 x 90 x 45 mm)(AS ONE,目录号:1-4698-09)
  11. 黑色96孔条板(Black Combiplate 8,Labsystems,目录号:95029450)
    注意:此产品已停产。
  12. Parafilm(Bemis,目录号:PM996)
  13. 1,2-二油酰 - sn - 甘油-3-磷酸胆碱(DOPC)(50毫克/毫升氯仿,Avanti Polar Lipids,目录号:850375C)
    注意:储存在-30°C。
  14. 来自鸡蛋的SM(Avanti Polar Lipids,目录号:860061P)
    注意:储存在-30°C&nbsp;
  15. 胆固醇(Sigma-Aldrich,目录号:C8667)
    注意:储存在-30°C。
  16. L-α-磷脂酰乙醇胺-N-(丽丝胺罗丹明B磺酰基)(铵盐)(Rhod PE)(氯仿中1mg / ml,Avanti Polar Lipids,目录号:810146C)
    注意:将溶液转移到带螺帽的圆底玻璃管中。用封口膜密封盖子以避免蒸发。避光,在-30°C下储存。用主要物质的分子量(1275.678g / mol)计算的近似摩尔浓度:0.8mM。
  17. 氯仿(Wako Pure Chemical Industries,目录号:038-02601)
    注意:氯仿具有挥发性和肝毒性。因此,当使用氯仿及其混合物时,必须在通风橱中使用它们或使用替代设备来处理化学品安全。
  18. 甲醇(Sigma-Aldrich,目录号:19-2410-4)
  19. 乙醇(Wako Pure Chemical Industries,目录号:057-00456)
  20. 水(超纯水,例如,Milli Q)
  21. 用于血细胞凝集抑制试验的UV灭活RuV颗粒(RuV抗原)(Denka Seiken,目录号:310071)
    注意:储存在-30°C。用1 ml /小瓶水重建后,在4°C下储存并在3天内使用。&nbsp;
  22. 氯化钙二水合物(CaCl 2 •2H 2 O)(NACALAI TESQUE,目录号:06731-05)
  23. Tris缓冲盐水(10x,pH 7.4)(TBS)(NACALAI TESQUE,目录号:35438-81)
  24. 用于哺乳动物细胞和组织提取物的蛋白酶抑制剂混合物(100x)(NACALAI TESQUE,目录号:25955-11)
  25. 5-(N-2,3-二羟基丙基乙酰氨基)-2,4,6-三碘-N,N'-双(2,3-二羟基丙基)间苯二甲酰胺(Nycodenz)(Sigma-Aldrich,目录号:D2158)
  26. 十二烷基硫酸钠(SDS)(NACALAI TESQUE,目录号:02873-75)
  27. 甘油(Wako Pure Chemical Industries,目录号:075-00616)
  28. 二硫苏糖醇(DTT)(NACALAI TESQUE,目录号:14112-94)
  29. Bromophenol blue(Wako Pure Chemical Industries,目录号:029-02912)
  30. 三(羟甲基)氨基甲烷(Tris)(NACALAI TESQUE,目录号:35434-21)
  31. 甘氨酸(NACALAI TESQUE,目录号:17109-64)
  32. 盐酸(HCl)(Wako Pure Chemical Industries,目录号:080-01066)
  33. 30%(w / v) - 丙烯酰胺/双混合溶液(37.5:1)(NACALAI TESQUE,目录号:06144-05)
  34. 过氧二硫酸铵(APS)(NACALAI TESQUE,目录号:02627-34)
  35. N,N,N',N'-四甲基乙二胺(TEMED)(NACALAI TESQUE,目录号:33401-72)
  36. 预染蛋白质标记物如PINK预染色蛋白质梯子(NIPPON Genetics,目录号:MWP02)和ExcelBand TM 3色常规范围蛋白质制造商(SMOBIO Technology,目录号:PM2500)
  37. 脱脂牛奶(BD,Difco,目录号:232100)
  38. 聚氧乙烯(20)脱水山梨糖醇单月桂酸酯(吐温20)(Wako Pure Chemical Industries,目录号:167-11515)
  39. 山羊抗RuV病毒粒子(株HPV-77)多克隆抗体(Acris Antibodies,目录号:BP1061)
    注意:储存在4°C。长时间储存,等分并储存在-30°C。
  40. 小鼠单克隆抗山羊/绵羊IgG-过氧化物酶抗体(Sigma-Aldrich,目录编号:A9452)
    注意:储存在4°C。对于长时间储存,等分并储存在-30°C。
  41. Immobilon Western Chemiluminescent HRP Substrate(Merck,目录号:WBKLS0500)
  42. 氯仿/甲醇(19/1,v / v)
  43. 0.2M CaCl 2
  44. 1 M DTT
  45. 在50%(v / v)乙醇中的1%(w / v)溴酚蓝
  46. 10%(w / v)SDS
  47. 1M Tris-HCl pH 8.8
  48. 1M Tris-HCl pH 6.7
  49. 10%(w / v)APS
  50. 20%(w / v)Tween 20
    注意:储存在4°C。使用前检查细菌或真菌污染。
  51. 脂质原液
  52. TBS(见食谱4)
  53. 2x TBS(见食谱5)
  54. 80%(w / v)Nycodenz / TBS(见食谱6)
  55. 30%(w / v)Nycodenz / TBS(见食谱7)
  56. 3x SDS样品缓冲液(见配方8)
    注意:分装并储存在-30°C&nbsp;
  57. 10%(w / v)聚丙烯酰胺分离凝胶溶液(见配方9)
  58. 5%(w / v)聚丙烯酰胺堆积凝胶溶液(见配方10)
  59. 10x Tris / Glycine(见食谱11)
  60. 电极缓冲液(见配方12)
  61. 转移缓冲区(见食谱13)
  62. TBS-T(见食谱14)
  63. 5%(w / v)脱脂牛奶/ TBS-T(见食谱15)
  64. 2%(w / v)脱脂牛奶/ TBS-T(见食谱16)

设备

  1. 微量移液器耐有机溶剂分配(NICHIRYO,型号:Nichipet EX Plus II,目录号:00-NPLO2-20,00-NPLO2-200,00-NPLO2-1000)&nbsp;
  2. Pipet-Aid XP移液器控制器(Drummond Scientific,目录号:4-040-101-J)
  3. 涡旋混合器(Delta混合器,TAITEC,型号:Se-08)
  4. 带水浴的氮气蒸发器(Nakajima seisakusho,Co.,Ltd.,定制)
  5. 通风柜配有活性炭过滤网(道尔顿,型号:DC-183-100)&nbsp;
  6. 探头式超声波处理器(Hielscher Ultrasonics,型号:UP50H)
  7. 微型冷冻离心机(例如,KUBOTA,型号:3520)
  8. 电子天平(Shimadzu,LIBROR,型号:EB-340HW; Mettler-Toledo International,型号:ML802 / 52)
  9. 超速离心机(Beckman Coulter,型号:Optima TM TLX,带TLS-55转子)
  10. 用于微量离心管的块加热器(Nippon Genetics,Fast Gene TM ,型号:FG-02N)
  11. pH计(TOA Electronics,型号:HM-30S)&nbsp;
  12. 微波炉(TOSHIBA,型号:ER-225)
  13. 用于SDS-PAGE的蛋白质电泳装置(例如,BIO CRAFT,型号:BE-S28用于宽微型凝胶)
  14. Western印迹装置(例如,Bio-Rad Laboratories,Criterion TM 印迹用于宽微型凝胶,目录号:1704070)
  15. 电源(ATTO,型号:AE-8450,用于SDS-PAGE; Bio-Rad Laboratories,型号:250 / 2.5,用于Western印迹)
  16. 管旋转器(SCINICS,型号:RVM-101)
  17. 互惠(TAITEC,型号:Personal-11)和/或跷跷板(TAITEC,型号:Wave-SI)振动器
  18. 化学发光成像系统(ATTO,型号:WSE-6200H LuminoGraph II)
  19. 微孔板读板机(BMG LABTECH,型号:FLUOstar Optima)

软件

  1. ImageSaver 6 for Windows(ATTO Co.)作为WSE-6200H LuminoGraph II的图像采集软件
  2. 用于Windows的CS Analyzer 4(ATTO Co.)作为图像分析软件
  3. Microsoft Excel 2016(Microsoft Corp.)作为电子表格软件
  4. GraphPad Prism 7(Graph Pad Software)作为图形绘制软件

程序

  1. 脂质体的制备
    除非另有说明,否则在室温下进行所有操作。当使用含有荧光标记的脂质的样品时,在整个过程中尽可能保护它们免受直射光的影响。
    1. 为了制备由DOPC / SM /胆固醇/ Rhod PE以8:2:3:0.05的摩尔比组成的2x浓缩脂质体,制备每种脂质在有机溶剂中的储备溶液,然后在适当体积的每种储备溶液中混合。圆底玻璃管,以获得最终量:1.6μmolDOPC,0.4μmolSM,0.6μmol胆固醇和10nmol Rhod PE(图1A)。对于其他组合物的脂质体,适当改变放入玻璃管中的脂质量(表1)。

      表1.2x浓缩脂质体的脂质组合物。每种脂质体含有等量的磷脂。 DOPC用作基质磷脂,因为磷脂酰胆碱是质膜中最丰富的磷脂类型。脂质体A(8/2/3)的DOPC / SM /胆固醇的摩尔比与先前关于鞘磷脂特异性毒素溶素的研究中使用的脂质体的摩尔比相同(Ishitsuka和Kobayashi,2007)。脂质体A和A'的组成相同。&nbsp;

      a 溶于氯仿/甲醇(19/1,v / v); b 溶于氯仿;蒸发后,每个玻璃管加入1毫升 c 。

    2. 使用设置在通风橱中的氮气蒸发器在氮气下在浴温35-40℃下完全蒸发有机溶剂。蒸发至少20分钟后,在玻璃管的底部形成脂质膜(图1B)。
    3. 用1ml TBS-Ca将所得脂质膜再水化(图1C)。要制备不含钙离子的脂质体,请使用TBS代替TBS-Ca。


      图1.脂质体的制备。 A.蒸发前脂质溶液的混合物; B.蒸发后形成的脂质膜; C.用TBS-Ca再水化的脂质膜; D.超声处理后的脂质悬浮。

    4. 用探针型超声波仪在混合水(无冰)中冷却,将混合物超声处理20-30分钟。对于UP50H超声波处理器,设置幅度为80%,占空比为50%。
      注意:混合物在开始时看起来很混浊。在30分钟(对于脂质体A)或20分钟(对于具有其他组合物的脂质体)超声处理后,混合物变得透明。每10分钟超声处理使管子涡旋,以恢复溅在管子内壁上的液体。
    5. 将脂质悬浮液转移至1.5ml微量离心管中。
    6. 使用微量冷冻离心机在25℃下以15,000 离心10分钟离心脂质悬浮液,以从超声波尖端沉淀脂质聚集体和碎片。
    7. 将上清液转移到新的1.5ml微量离心管中,并在相同条件下再次离心。重复离心直至形成不可见的颗粒。
    8. 将上清液转移到新的1.5ml微量离心管中,并将该级分作为2x浓缩的脂质体级分在4℃下储存。避光,3天内使用。

  2. Nycodenz密度梯度离心
    使用Nycodenz密度梯度分级评估RuV抗原和脂质体之间的相互作用。该实验的示意图如图2所示。除非另有说明,否则在室温下进行所有操作。使用前将所有试剂加热至室温。要省略每次反应中的钙离子,请使用基于TBS的试剂代替基于TBS-Ca的试剂。


    图2. Nycodenz密度梯度分级的示意图

    1. 将以下组分混合在聚碳酸酯超速离心管中:
      42μl水
      75μl2xTBS-Ca
      3μl100x蛋白酶抑制剂鸡尾酒
      30μlRuV抗原
      (总体积:150μl)
      注意:&nbsp;
      1. RuV抗原必须最后加入。在添加RuV抗原之前和之后通过移液充分混合。&nbsp;
      2. RuV抗原含有由制造商作为添加剂添加的牛血清白蛋白。它的浓度没有透露。
    2. 将步骤A8中制备的150μl2x浓缩脂质体级分加入混合物中,并通过移液管充分混合。
    3. 将混合物孵育1小时,避光。
    4. 通过移液将反应混合物(300μl)与300μl80%(w / v)Nycodenz / TBS-Ca混合以获得均匀溶液。所得溶液含有40%(w / v)Nycodenz。
    5. 用300μl30%(w / v)Nycodenz / TBS-Ca覆盖40%(w / v)Nycodenz溶液,小心不要将它们混合。
    6. 用300μlTBS-Ca覆盖30%(w / v)Nycodenz溶液,注意不要将它们混合。
    7. 称重每个管和摆动桶。通过在管中加入TBS-Ca,将两组管子和桶的平衡重量放在TLS-55转子中,以0.01克的比例对角放置。
      注意:虽然铲斗的重量最初是由制造商匹配的,但铲斗需要在重量上保持平衡,因为它们会随着时间的推移而磨损。
    8. 将每根管子小心地放入摆动桶中。
    9. 小心地将水桶吊入TLS-55转子,然后将转子安装在Optima TLX超速离心机中。
    10. 将样品以55,000rpm(259,000 x g max )在25℃下离心5小时。
    11. 小心地从铲斗上取下每根管子。
    12. 使用具有1-200μl尖端的P-200微量移液管从梯度顶部收集50μl等分试样。始终将移液管尖端的点设置在水面下方(≤1mm),并以圆周运动缓慢吸取溶液,小心不要吸入空气。&nbsp;
    13. 将两个50μl等分试样在1.5ml微量离心管中混合(即,~100μl/管)。尽管可以使用微量移液管同时取100μl等分试样,但作者建议使用50μl等分试样:更容易控制短移液冲程,从而降低干扰梯度的风险。将每个样品分级成12个级分。当最后一部分的体积小于100μl时,用TBS-Ca将体积调节至~100μl。
      注意:事先使用100μl水,在1.5 ml管上标记约100μl线用于最后一部分。
    14. 将30μl每种级分转移到1.5ml微量离心管中,然后与15μl3xSDS样品缓冲液混合。储存在-30℃直至SDS-PAGE分析。 15μl所得溶液含有10%(10μl)的每种收集的级分。
    15. 用56μlTBS-Ca稀释24μlRuV抗原,然后与40μl3xSDS样品缓冲液混合。储存在-30℃直至SDS-PAGE分析。 15μl所得溶液含有3μlRuV抗原,其对应于输入每个超速离心管的那些抗原的10%。将残余部分(约70μl/管)储存在4°C直至测量荧光。

  3. SDS-PAGE和Western印迹分析
    使用标准SDS-PAGE分析收集的级分中的RuV颗粒的分布,然后对RuV蛋白进行Western印迹分析。除非另有说明,否则在室温下进行所有操作。
    1. 参见SDS-PAGE的基本方案(He,2011)。根据制造商的说明,使用蛋白质电泳仪的凝胶浇铸装置制备10%(w / v)聚丙烯酰胺分离凝胶。&nbsp;
    2. 类似地,准备5%(w / v)聚丙烯酰胺堆积凝胶。
    3. 将凝胶装配到电泳装置中并倒入电极缓冲液。
    4. 使用块加热器在步骤B14和B15中在95℃下制备的用于SDS-PAGE分析的样品加热5分钟,然后冷却至室温。在25℃下以15,000 x g 旋转约10秒。
    5. 每个泳道加入15μl所得样品。类似地加载5μl预染色的分子标记物。
    6. 根据制造商的说明,以100-200 V常数进行电泳。
    7. 电泳后,将凝胶置于干净的容器中,并使用倒置或跷跷板振荡器在转移缓冲液中摇动10分钟。用甲醇预湿PVDF膜并类似地摇动它。
    8. 设置一层三明治,包括色谱纸,凝胶和膜,浸泡在转移缓冲液中。
    9. 将三明治放入装有转移缓冲液的Western印迹装置的腔室中。
    10. 根据制造商的说明,在4℃的冷室中以100V恒定电泳将蛋白质从凝胶转移至膜30分钟。
    11. 将膜置于干净的容器中,用5%(w / v)脱脂乳/ TBS-T浸泡1小时,同时摇动。
    12. 在TBS-T中将膜洗涤三次,每次5分钟。
    13. 将膜置于干净的容器中,加入1:4,000稀释的一抗(山羊抗RuV多克隆抗体)和2%(w / v)脱脂乳/ TBS-T。
    14. 将膜在4℃下孵育过夜,同时摇动。
    15. 在TBS-T中将膜洗涤三次,每次5分钟。
    16. 将膜置于干净的容器中,加入1:4,000稀释的二抗(HRP-缀合的小鼠抗山羊/绵羊IgG)和2%(w / v)脱脂乳/ TBS-T。
    17. 将膜孵育1小时,同时摇动。
    18. 在TBS-T中将膜洗涤三次,每次10分钟。
    19. 根据制造商的说明,通过将膜与Immobilon Western Chemiluminescent HRP底物或等效试剂一起孵育来产生信号。
    20. 根据制造商的说明,使用WSE-6200H LuminoGraph II图像分析仪检测信号。

  4. 荧光测量
    基于Rho PE的荧光强度分析收集的级分中脂质体的分布。在测量前将收集的级分和2x浓缩的脂质体级分加热至室温。
    1. 将50μl每种收集的级分(相当于每种级分的50%)和2x浓缩的脂质体级分(相当于总输入的33%)转移到96孔黑色微孔板的孔中。
    2. 使用FLUOstar Optima酶标仪在544 nm激发和终点模式下590 nm发射,根据制造商的说明测量每个孔中Rhod PE的荧光强度,无抖动功能。&nbsp;
    3. 以excel文件格式保存结果。

数据分析

  1. 使用图像采集和分析软件可视化收集的部分中的RuV蛋白。 RuV E1(两种包膜糖蛋白之一)和衣壳蛋白的表观分子量分别为58-65kDa和33-38kDa。图3显示了在各种脂质体存在或不存在下,Nycodenz密度梯度分级分离的E1和衣壳蛋白的分布。 RuV颗粒似乎存在于含有两种蛋白质的级分中。致密级分(级分10-12)仅含有衣壳蛋白,表明这些级分中不存在RuV颗粒。尽管在不存在脂质体的情况下RuV颗粒主要在级分6中检测到(图3F),但是在由DOPC / SM /胆固醇组成的脂质体存在下它们转变为级分4(图3A和3A')。当从反应中省略钙离子时,未观察到向较低密度的转变(图3E),表明RuV颗粒以钙依赖性方式与脂质膜相互作用,与先前的研究一致(Dubé等人< / em>,2014)。此外,当从脂质体中省略SM或胆固醇时,不再观察到这种转变(图3B和3C),并且使用仅由DOPC组成的脂质体(图3D),表明RuV颗粒与脂质膜之间的钙依赖性相互作用。在膜中需要SM和胆固醇。在图3中,显示了与原始文章(Otsuki et al。,2018)中描述的单独实验的结果。从三个独立的实验中获得了类似的结果。


    图3.在Nycodenz梯度上RuV蛋白的分布。 RuV抗原(紫外线灭活的RuV颗粒)在有或没有脂质体组合物的情况下,在钙的存在(+)或不存在( - )下孵育离子然后在Nycodenz梯度上进行脂质体浮选测定。通过蛋白质印迹分析检测每个级分中的病毒E1和衣壳(C)蛋白。除了图F之外,每个图的字母代码对应于表1中描述的脂质体的字母代码,其表示没有脂质体的结果。所示的值是每μlRuV抗原加入的每种脂质体级分中DOPC,SM和胆固醇(Chol。)的量(nmol)。所有脂质体还含有0.05nmol(每μlRuV抗原)的Rhod PE。星号表示牛血清白蛋白,它是RuV抗原的添加剂。显示了两组独立的离心实验:一组,上四组(A-D),另一组,下三组(A'-F)。在相同条件下(在高灵敏度模式下曝光10分钟)进行图像获取,并且将图像的对比度调整到相同的程度(将上限值设置为40,000并且将下限值设置为零)。图A和A'代表使用具有相同脂质组成和相同实验程序的脂质体的单独实验的结果。在该测定中,病毒蛋白的分布相似,但它们的总体信号强度在实验中变化。尽管整体信号强度不是该测定的主要问题,但这种可变性的可能原因是每种凝胶中的转移效率不稳定。

  2. 使用图像分析软件定量衣壳蛋白并确定每个级分中蛋白质的回收率。为此目的,E1蛋白不适合,因为作为RuV抗原的防腐剂添加的大量牛血清白蛋白在输入级分的蛋白质印迹上基本上与E1蛋白重叠。衣壳蛋白的总回收率在约10%至约70%之间变化,实验可能是由于Western印迹分析的半定量性质和/或在超速离心期间或之后蛋白质的降解。我们没有发现脂质体的类型与衣壳蛋白的回收之间存在任何相关性。
  3. 为了检查脂质体的分布,使用荧光强度数据计算在每个级分中回收的Rhod PE的%。图4显示了密度梯度级分中脂质体的分布。无论脂质组成如何,在顶部和第二部分中回收大部分脂质体(~70%或更多)。由于漂浮的脂质体不能完全回收,因此在整个分级过程中少量的它们(<10%)保留在水性表面上并在最后的部分中回收。在图4中,显示了与原始文章(Otsuki et al。,2018)中描述的单独实验的结果。从三个独立的实验中获得了类似的结果。


    图4.脂质体在Nycodenz梯度上的分布。使用Rhod PE的荧光强度监测脂质体的分布。数值表示为从梯度的每个部分回收的Rhod PE的总荧光强度的百分比(%)。字母代码对应于表1中描述的脂质体。

笔记

  1. Avanti Polar Lipids,Inc。( https://avantilipids.com/ )的主页为初学者提供脂质处理信息。
  2. Axis-Shield Density Gradient Media的主页,Alere Technologies AS的品牌( http://www.optiprep.com/)提供有关Nycodenz的一般信息。
  3. RuV颗粒的峰位置在实验之间大部分是可再现的,但是可以根据各个实验条件的微小差异而变化。因此,应直接比较待比较的样品,离心和分级。

食谱

注意:除非另有说明,以下配方中使用的水是超纯水。

  1. DOPC储备液(5 mg / ml)
    1. 将1ml DOPC(50mg / ml,在氯仿中)放入带有螺帽的圆底玻璃管中
    2. 用9毫升氯仿/甲醇(19/1,v / v)稀释
    3. 用封口膜密封盖子以减少溶剂的蒸发
    4. 储存于-30°C并在使用前温热至室温
    注意:为了获得DOPC和SM(见下文)储备溶液的摩尔浓度,根据Rouser等人确定溶液中磷的浓度。 (1966)。
  2. SM原液(5 mg / ml)
    1. 称取50毫克SM(粉末)并放入带有螺帽的圆底玻璃管中
    2. 将SM溶于10毫升氯仿/甲醇(19/1,v / v)中
    3. 用封口膜密封盖子以减少溶剂的蒸发
    4. 储存在-30°C。使用前温热至室温
  3. 胆固醇储备溶液(2mg / ml,计算的摩尔浓度:5.17mM)
    1. 称取20毫克胆固醇(粉末),放入带螺帽的圆底玻璃管中
    2. 将胆固醇溶于10毫升氯仿/甲醇(19/1,v / v)中
    3. 用封口膜密封盖子以减少溶剂的蒸发
    4. 储存在-30°C。使用前温热至室温
  4. TBS
    25mM Tris-HCl pH 7.4
    137 mM NaCl
    2.68 mM KCl
    1. 将2.5ml 10x TBS加入50ml离心管中
    2. 用水填充至25毫升并充分混合
    为了制备含有1 mM CaCl 2 (TBS-Ca)的TBS,在加水前加入125μl0.2M CaCl 2
    储存在4°C
  5. 2x TBS
    在2 ml微量离心管中用1.6 ml水稀释400μl10xTBS
    为了制备2x TBS-Ca,加入20μl0.2MCaCl 2 (终浓度:2mM)并充分混合
    储存在4°C
  6. 80%(w / v)Nycodenz / TBS
    1. 将8克Nycodenz用热水溶解在15毫升离心管中 注意:需要时间将少量Nycodenz溶解在少于10毫升的水中。使用通过微波预热的热水并密集混合。偶尔用热自来水加热管子。如果出现难以熔化的团块,请在浴式超声波仪中通过超声破碎它们。
    2. 加入1毫升10倍TBS,加水至10毫升。充分混合并在4°C下储存。
    3. 使用前,将适量的溶液转移到2.0毫升的微量离心管中,加入1/100体积的100倍蛋白酶抑制剂混合液。好好混合。
    为了制备80%(w / v)Nycodenz / TBS-Ca,还加入1/200体积的0.2M CaCl 2 (终浓度:1mM)并充分混合
  7. 30%(w / v)Nycodenz / TBS
    1. 将3克Nycodenz溶解在15毫升离心管中的热水中
    2. 加入1毫升10倍TBS
    3. 用水填充至10毫升并充分混合。储存在4°C
    4. 使用前,将适量的溶液转移到2.0毫升的微量离心管中,加入1/100体积的100倍蛋白酶抑制剂混合液。好好混合
    为了制备30%(w / v)Nycodenz / TBS-Ca,还加入1/200体积的0.2M CaCl 2 (终浓度:1mM)并充分混合
  8. 3x SDS样品缓冲液
    1. 将以下组分混合在15毫升离心管中:
      4.5毫升甘油(终浓度:30%(v / v))
      2.25毫升1M DTT(终浓度:150 mM)
      在50%(v / v)乙醇中加入1.5ml 1%(w / v)溴酚蓝(终浓度:0.1%[w / v])
      2.81毫升1M Tris-HCl pH 6.7(终浓度:187.5 mM)
    2. 使用管旋转器溶解0.9g SDS(终浓度:6%[w / v])
    3. 用水补充至15毫升并充分混合
    4. 储存于-30°C并在37°C温热,以在使用前溶解SDS
  9. 10%(w / v)聚丙烯酰胺分离凝胶溶液
    1. 将以下组分混合在50毫升离心管中:
      8.28毫升水
      11.25ml 1M Tris-HCl pH 8.8(终浓度:375mM)
      10毫升30%(w / v) - 丙烯酰胺/双混合溶液
      300μl10%(w / v)SDS(终浓度:0.1%[w / v])
      300μl10%(w / v)APS(终浓度:0.1%[w / v])
    2. 加入15μlTEMED(终浓度:0.05%[v / v])并充分混合
    3. 立即倒入凝胶铸造设备
    注意:这个体积足以容纳两个宽的迷你凝胶。
  10. 5%(w / v)聚丙烯酰胺堆积凝胶溶液
    1. 将以下组分混合在15毫升离心管中:
      6.92毫升水
      1.25ml 1M Tris-HCl pH 6.7(终浓度:125mM)
      1.67毫升30%(w / v) - 丙烯酰胺/双混合溶液
      100μl10%(w / v)SDS(终浓度:0.1%[w / v])
      100μl10%(w / v)APS(终浓度:0.1%[w / v])
    2. 加入10μlTEMED(终浓度:0.1%[v / v])并充分混合
    3. 立即浇铸在分离凝胶上
    注意:此体积足以容纳两个宽的迷你凝胶。
  11. 10x Tris / Glycine
    1. 将121g Tris和576g甘氨酸溶于约3L水中
    2. 用水充满4升并充分混合
    3. 在室温下储存
  12. 电极缓冲器
    1. 用约800毫升水稀释100毫升10倍Tris /甘氨酸
    2. 加入10毫升10%(w / v)SDS
    3. 用水补充至1升并充分混合
    4. 在室温下储存
  13. 转移缓冲区
    1. 用1.4L水稀释200ml 10x Tris /甘氨酸
    2. 加入400毫升甲醇并充分混合
    3. 保持在冰上或4°C
    注意:加水前不要将甲醇加入10x Tris /甘氨酸中。混合物变混浊。
  14. TBS-T
    1. 用少于900毫升的水稀释100毫升10倍TBS
    2. 加入5毫升20%(w / v)吐温20,使最终浓度为0.1%(w / v)
    3. 用水补充至1升并充分混合
    4. 储存于4°C并在使用前温热至室温
  15. 5%(w / v)脱脂牛奶/ TBS-T
    使用管式旋转器将2.5 g脱脂牛奶溶解在50 ml离心管中的50 ml TBS-T中
    注意:将管混合至少1小时以完全溶解。在使用当天做好准备,并在4°C下储存。在2天内使用。
  16. 2%(w / v)脱脂牛奶/ TBS-T
    用管式旋转器将1克脱脂牛奶溶解在50毫升TBS-T的50毫升离心管中。
    注意:将管混合至少1小时以完全溶解。在使用当天做好准备,并在4°C下储存。在2天内使用。

致谢

该协议已在Otsuki et al。,2018中使用。该研究得到了AMED-CREST(日本医学研究与开发机构,进化科学与技术核心研究机构)的KH资助,授权号:JP18gm0910005)。该协议改编自Busse 等人的工作(2013)。作者声明他们没有利益冲突。

参考

  1. Busse,R.A.,Scacioc,A.,Hernandez,J.M.,Krick,R.,Stephan,M.,Janshoff,A.,Thumm,M。和Kuhnel,K。(2013)。 基于脂质体的方法对蛋白质 - 磷酸肌醇相互作用的定性和定量表征。 自噬 9(5):770-777。&nbsp;
  2. Dubé,M.,Rey,F。A.和Kielian,M。(2014)。 风疹病毒:第一种需要钙的病毒融合蛋白。 PLoS Pathog < / em> 10(12):e1004530。&nbsp;
  3. DuBois,R.M.,Vaney,M.C.,Tortorici,M.A.,Kurdi,R.A.,Barba-Spaeth,G.,Krey,T。和Rey,F.A。(2013)。 风疹病毒蛋白E1晶体结构的功能和进化见解。 自然 493(7433):552-556。
  4. 他,F。(2011)。 Laemmli-SDS-PAGE。 Bio-protocol Bio101 :e80。&nbsp。 ;
  5. Hobman,T。C.(2013)。风疹病毒。在:Knipe,D.M.,Howley,P.M。,Cohen,J.I.,Griffin,D.E.,Lamb,R.A.,Martin,M.A.,Racaniello,V.R.,Roizman,B.(Eds。)。 菲尔兹病毒学。第6版。第1卷,第687-711页。 Lippincott Williams&amp;威尔金斯,费城,宾夕法尼亚州。
  6. Ishitsuka,R。和Kobayashi,T。(2007)。 胆固醇和脂质/蛋白质比例可控制鞘磷脂特异性毒素,lysenin的寡聚化。 生物化学 46(6):1495-1502。&nbsp;
  7. Mastromarino,P.,Cioè,L.,Rieti,S。和Orsi,N。(1990)。 膜磷脂和糖脂在风疹病毒Vero细胞表面受体中的作用。 Med Microbiol Immunol 179(2):105-114。&nbsp;
  8. Mastromarino,P.,Rieti,S.,Cioè,L。和Orsi,N。(1989)。 风疹病毒在红细胞膜上的结合位点。 Arch Virol 107(1-2):15-26。&nbsp;
  9. Otsuki,N.,Sakata,M.,Saito,K.,Okamoto,K.,Mori,Y.,Hanada,K。和Takeda,M。(2018)。 宿主细胞膜中的鞘磷脂和胆固醇都是风疹病毒进入的必要条件。 J Virol。 92(1):e01130-17。
  10. Rouser,G.,Siakotos,A。N.和Fleischer,S。(1966)。 通过薄层色谱和斑点磷分析对磷脂进行定量分析。 脂质 1(1):85-86。&nbsp;
  11. Zhao,H。和Lappalainen,P。(2012)。 分析蛋白质 - 脂质相互作用的生化方法的简单指南。 Mol Biol Cell 23(15):2823-2830。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Saito, K., Otsuki, N., Takeda, M. and Hanada, K. (2018). Liposome Flotation Assay for Studying Interactions Between Rubella Virus Particles and Lipid Membranes. Bio-protocol 8(16): e2983. DOI: 10.21769/BioProtoc.2983.
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