搜索

Immunoprecipitation of Cell Surface Proteins from Gram-negative Bacteria
革兰氏阴性菌细胞表面蛋白的免疫沉淀   

下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

The meningococcus (Neisseria meningitidis) remains an important threat to human health worldwide. This Gram-negative bacterium causes elevated disabilities and mortality in infected individuals. Despite several available vaccines, currently there is no universal vaccine against all circulating meningococcal strains (Vogel et al., 2013). Herein, we describe a new protocol that is capable of identifying only cell surface exposed proteins that play a role in immunity, providing this research field with a more straightforward approach to identify novel vaccine targets. Even though N. meningitidis is used as a model in the protocol herein described, this protocol can be used for any Gram-negative bacteria provided modifications and optimizations are carried out to adapt it to different bacterial and disease characteristics (e.g., membrane fragility, growth methods, serum antibody levels, etc.).

Keywords: Gram-negative(革兰氏阴性), Immunoproteome(免疫蛋白质组), Immunoprecipitation(免疫沉淀), Cell surface antigen(细胞表面抗原), Outer membrane protein(外膜蛋白), Exposed antigen(暴露的抗原)

Background

Attempts to develop novel vaccines against N. meningitidis often rely on 2D SDS-PAGE (two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and Western blot followed by MS (mass spectrometry) (Wheeler et al., 2007). However, such approach employs whole cell lysate, identifying a plethora of proteins that do not have vaccine potential (Mendum et al., 2009). We therefore aimed at developing a method capable of identifying only cell surface exposed proteins that might play important role in immunity. Briefly, our protocol consists in growing the pathogen of interest, immunoprecipitating surface antigens with sera of immune individuals, and identifying immunoprecipitated proteins by liquid chromatography-tandem mass spectrometry. We were able to identify 23 meningococcal surface antigens using this new protocol, some of which are components of commercially available vaccines (Newcombe et al., 2014). We also have adapted this protocol to other Gram-negative bacteria and have obtained promising results: we identified previously described surface-exposed proteins, many of which have already been tested as vaccine or diagnostic test candidates. These results show this is a robust technique that can be applied to a diverse range of Gram-negative bacteria and capable of yielding high-quality results that can be further exploited by a myriad of applications (e.g., vaccines, diagnosis, etc.).

Materials and Reagents

  1. Disposable Petri dishes (Cromwell Group, catalog number: STS3855002B )
  2. L-shaped cell spreaders (Fisher Scientific, catalog number: 14-665-231 )
  3. Disposable inoculating loop (Sigma-Aldrich, catalog number: I8388 )
  4. 1.5 ml microcentrifuge tubes (Corning, Axygen®, catalog number: MCT-150-C )
  5. Protein LoBind tube (Eppendorf, catalog number: 022431102 )
  6. 20 ml plastic universals (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 128BFS )
  7. Plastic sealable bags (Fisher Scientific, catalog number: 10366984)
    Manufacturer: MINIGRIP, catalog number: BAJ-340-091N .
  8. PierceTM spin columns (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 69725 )
  9. Disposable sterile scalpel No. 10 (WMS, catalog number: W259 )
  10. 10 µl Filter tips (STARLAB INTERNATIONAL, TipOne®, catalog number: S1121-3810 )
  11. 20 µl Filter tips (STARLAB INTERNATIONAL, TipOne®, catalog number: S1120-1810 )
  12. 200 µl Filter tips (STARLAB INTERNATIONAL, TipOne®, catalog number: S1120-8810 )
  13. 1,000 µl Filter tips (STARLAB INTERNATIONAL, TipOne®, catalog number: S1122-1830 )
  14. Neisseria meningitidis (strains L9153 and MC58, Public Health England)
  15. Phosphate-buffered saline (PBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10010001 )
  16. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A9418 )
  17. Human serum (Sigma-Aldrich, catalog number: H6914 )
  18. Disease state serum (acquisition of this varies and depends on the pathogen being investigated)
  19. PierceTM Protein A/G UltraLinkTM Resin (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 53132 )
  20. SeeBlue® protein marker (Thermo Fisher Scientific, NovexTM, catalog number: LC5925 )
  21. Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: 43815 )
  22. Iodoacetamide (Sigma-Aldrich, catalog number: I6125 )
  23. InvitrogenTM NovexTM NuPAGETM 10x sample reducing agent (Thermo Fisher Scientific, NovexTM, catalog number: NP0004 )
  24. InvitrogenTM NovexTM NuPAGETM 4x LDS loading buffer (Thermo Fisher Scientific, NovexTM, catalog number: NP0007 )
  25. InvitrogenTM NovexTM NuPAGETM 12% Bis-Tris 1 mm–10 wells (Thermo Fisher Scientific, InvitrogenTM, catalog number: NP0341BOX )
  26. InvitrogenTM NovexTM NuPAGETM MOPS running buffer 20x (Thermo Fisher Scientific, NovexTM, catalog number: NP000102 )
  27. SimplyBlueTM Safe Stain (Thermo Fisher Scientific, NovexTM, catalog number: LC6060 )
  28. Acetonitrile for HPLC-MS (Fisher Scientific, catalog number: 10616653 )
  29. Ammonium bicarbonate (Sigma-Aldrich, catalog number: 09830 )
  30. Trypsin Gold, mass spectrometry grade (Promega, catalog number: V5280 )
  31. Columbia blood agar base (Oxoid, catalog number: CM0331 )
  32. Horse blood, defibrinated (Oxoid, catalog number: SR0050 )
  33. Tris base (Roche Diagnostics, catalog number: 10708976001 )
  34. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9625 )
  35. EDTA (Sigma-Aldrich, catalog number: E6758 )
  36. Triton X-100 (Sigma-Aldrich, catalog number: X100 )
  37. Sodium deoxycholate (Sigma-Aldrich, catalog number: 30970 )
  38. Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L3771 )
  39. Formic acid LC/MS (Fisher Scientific, catalog number: A117-50 )
  40. Water for HPLC-MS (Fisher Scientific, catalog number: 10777404 )
  41. Methanol (Sigma-Aldrich, catalog number: 179957-1L )
  42. Acetic acid (Sigma-Aldrich, catalog number: A6283 )
  43. CAB medium (see Recipes)
  44. PBS plus 1% BSA (see Recipes)
  45. Solubilization buffer (see Recipes)
  46. 1% SDS (see Recipes)
  47. 1 M dithiothreitol stock solution (see Recipes)
  48. 1 M Iodoacetamide (see Recipes)
  49. 25 mM ammonium bicarbonate (see Recipes)
  50. Acetonitrile (50% v/v) in 25 mM ammonium bicarbonate (see Recipes)
  51. Acetonitrile (50% v/v) (see Recipes)
  52. Solvent A for HPLC (see Recipes)
  53. Solvent B for HPLC (see Recipes)
  54. 0.1% formic acid/50% acetonitrile solution (see Recipes)

Equipment

  1. Gilson Pipette Pipetman Classic P2 (Gilson, catalog number: F144801 )
  2. Gilson Pipette Pipetman Classic P20 (Gilson, catalog number: F123600 )
  3. Gilson Pipette Pipetman Classic P200 (Gilson, catalog number: F123601 )
  4. Gilson Pipette Pipetman Classic P1000 (Gilson, catalog number: F123602 )
  5. Eppendorf® Micro centrifuge 5415R (Eppendorf, model: 5415 R )
  6. BOD low temperature refrigerated incubator (VWR, model: BOD Incubator 2005 , catalog number: 35960-056)
  7. CO2 incubator (LEEC, model: Culture Safe Precision 190D )
  8. Stuart Gyratory rocker (Cole-Parmer, Stuart, model: SSL3 )
  9. Vortex Mixer SA8 (Cole-Parmer, Stuart, model: SA8 )
  10. Ultrasonic bath (Fisher Scientific, model: FisherbrandTM S-Series , catalog number: 10611983)
  11. Ultimate 3000 nano HPLC system (Thermo Fisher Scientific, Thermo ScientificTM, model: UltiMateTM 3000 RSL Cnano System )
  12. AcclaimTM C-18 PepmapTM column (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 164569 )
  13. QSTAR® XL hybrid LC/MS/MS system (Applied Biosystems, model: QSTAR® XL Hybrid LC/MS/MS System )
  14. Eppendorf MixMateTM (Fisher Scientific, catalog number: 21-379-00)
    Manufacture: Eppendorf, catalog number: 022674200 .
  15. Eppendorf ThermoStat Plus interchangeable block heater (Eppendorf, model: ThermoStat Plus , catalog number: 022670204)
  16. Eppendorf VacufugeTM concentrator (Eppendorf, model: VacufugeTM Concentrator , catalog number: 022820001)
  17. Heraeus Tube roller Spiramix (Fisher Scientific, catalog number: 10600653)
    Manufacturer: Thermo Fisher Scientific, Thermo ScientificTM, catalog number: DS507 .
  18. Fume hood

Software

  1. Chromeleon v6.8 or later (Dionex, Thermo Fisher Scientific)
  2. Analyst QS 1.1 (Applied Biosystems)
  3. Mascot v2.2.04 or later (Matrix Science, London)

Procedure

  1. Cell surface immunoprecipitation
    1. Grow N. meningitidis from frozen stock: Obtain a small amount of frozen culture with a sterile 200 µl tip, inoculate Columbia agar base (CAB) plate and spread to give single colonies. Incubate overnight (~18 h) at 35 °C with 5% CO2. Collect 20-30 colonies and spread over the entire surface of a CAB plate, using a sterile loop, and incubate for 4 h at 35 °C with 5% CO2. Individual colonies will not be seen at this stage.
    2. Recover bacteria by adding PBS to the plate and mixing the colonies into the PBS using a sterile loop. Remove the PBS/bacteria suspension using a P1000 micropipette (1 ml per plate, use half plate for each immunoprecipitation), centrifuge for 2 min at 13,000 x g at room temperature, Wash pellet three times with 1 volume PBS plus 1% BSA. For these and subsequent wash steps, pellet cells by centrifugation for 2 min at 13,000 x g at room temperature, remove supernatant, add fresh buffer and resuspend the pellet.
      Notes:
      1. If using liquid culture instead of plates, we suggest harvesting cells at mid-log growth phase and conditions that favor the expression of virulence factors. We suggest researchers do a growth curve with their organism of interest or have good knowledge of how the organism grows in liquid media to be able to properly identify growth phases.
      2. Centrifugation conditions may vary according to the bacterium in question. The presence of lysed cells will increase the selection on non-surface proteins.
      3. Make every effort to ensure that early centrifugation steps will not disrupt cells while removing secreted proteins or components of lysed, dead cells (see Note 2).
    3. Suspend pellet from previous step in 1 ml of serum sample (control or test [items 17 and 18 of Materials and Reagents list, respectively]) and rotate for 90 min at 4 °C.
      Note: Proteins identified in the control serum samples should be disregarded in future analysis as they are likely to play no role in the immune response against the pathogen. Researchers should make sure the control sera they have is indeed negative against for the pathogen they are investigating.
    4. Pellet the cells (13,000 x g, 5 min, RT) and wash twice with PBS plus 1% BSA.
    5. Resuspend cells in 1 ml solubilization buffer and incubate for 60 min at 37 °C.
    6. Centrifuge sample (13,000 x g, 60 min, RT), collect supernatant and incubate with protein A/G UltraLink beads (50 μl) for 18 h at 4 °C with agitation or rolling.
    7. Centrifuge sample (2,500 x g, 2 min, RT), collect beads.
    8. Wash beads five times (2,500 x g, 2 min) with 1 volume solubilization buffer.
    9. Elute antibody:antigen complex with 1 volume SDS (1%) and boiling (10 min).
    10. Add 4 volumes of water and vortex to elute proteins. Centrifuge (2,500 x g, 3 min, RT), and collect supernatant; the increase in volume allows for a more efficient removal of proteins during vortexing.
    11. Concentrate sample back to 50 μl so SDS concentration is back to 1% (approximately 45 min at 45 °C using Speed-Vac). At this point, the samples can be mixed with loading buffer and run on an SDS PAGE gel. Proceed to step B1. If the samples are being prepared for mass spectrometry analysis, the proteins can be reduced and alkylated prior to separation. Reduce the eluted proteins by adding DTT to a final concentration of 10 mM and boil for 2 min (for 50 μl, add 0.5 μl of 1 M DTT stock solution).
    12. Add iodoacetamide to a final concentration of 50 mM (for 50 μl, add 2.5 μl of 1 M IAA stock solution) and incubate for 30 min at room temperature in the dark.

  2. 1D SDS-PAGE and sample digestion
    1. Mix reduced, alkylated samples with loading buffer (item 23 of Materials and Reagents list) and boil for 5 min.
    2. Separate proteins by SDS-PAGE (12%, 1 mm thick, 10 cm long).
    3. Stain with SimplyBlueTM for 1 h and de-stain with ultra-pure water (according to manufacturer’s instructions–see Figure 1).


      Figure 1. Representative image of immunoprecipitated (IP) surface proteins of the  meningococcus. Reproduced from Newcombe et al. (2014).

    4. Cut gel lanes in as many slices as necessary based on sample protein concentration and place slices in low protein bind tubes.
      Note: A full gel lane can be cut into 20 slices, however, it may only be necessary to run a shortened gel and cutting it into 5 slices.
    5. Wash gel slices with 50% acetonitrile in 25 mM ammonium bicarbonate twice with vortexing for 15 min at room temperature to remove stain–use enough volume to cover the gel slices.
    6. Dehydrate gel slice by incubation with 100% acetonitrile (20 min, room temperature).
    7. Dry gel pieces using a Speed-Vac to dryness (about 20 min).
    8. Rehydrate in 25 mM ammonium bicarbonate containing modified trypsin (12.5 ng/μl–the final volume will depend on the size of the gel slice, 4 °C for 10 min). If required add more 25 mM ammonium bicarbonate to cover gel pieces, incubate overnight at 37 °C.
    9. Remove digest solution, extract digested proteins by incubation with 30 μl 0.1% formic acid/50% acetonitrile for 20-30 min with vortexing followed by 5 min in a sonicating bath (40 kHz)–repeat once and collect supernatant each time.
    10. Completely dry extracted digested proteins using Speed-Vac and resuspend in 20 μl water. Store at -20 °C.

  3. Liquid chromatography-electrospary ionization-MS/MS (please see Notes)
    1. Separate peptides using Ultimate 3000 HPLC and C18 pepman (Dionex) column at 40 °C, 350 nl/min, gradient of 2-50% solvent B for 30 min, 90% solvent B for 5 min–controlled by Chromeleon software.
    2. Set QSTAR XL to 2.3 kV electrospray, curtain gas at 8 arbitrary units, and 1 sec survey from 400 to 1,200 m/z with charge states 2-4.
    3. Acquire MS data with Information Data Acquisition with Analyst QS 1.1 software.
    4. Select most intense product ions with m/z of between 65 and 1,200 amu for MS/MS.

  4. Database analysis (please see Notes)
    1. Submit peak lists of MS/MS spectra to Mascot Server (v2.2.04; Matrix Science) and analyse using the MS/MS Ions search programme.
    2. Allow up to one missing trypsin cleavage with fixed modification of carbamidomethyl (C) and variable modification of oxidation (M).
    3. Set peptide and MS/MS fragment tolerance to 1.2 and 0.6 Da respectively and select MH22+ and MH33+ as the precursor charge states.
    4. Search identified proteins against the National Center for Biotechnology Information nonredundant (NCBInr) database with the taxonomy set N. meningitidis (taxonomy code 487). Analyse the data with the appropriate database for the organism being used.

Data analysis

Once a list of identified proteins is obtained it should be checked to ensure all proteins are surface exposed. This can be done using cellular localization prediction tools such as PSORTb v3.0 (Yu et al., 2010), CELLO v2.5 (Yu et al., 2004 and 2006) or Gneg-mPLoc (Shen and Chou, 2010). Searching for signal peptides or lipidation can also be useful. We recommend the following tools: LipoP 1.0 (Juncker, 2003), SignalP 4.1 (Petersen et al., 2011), Signal-CF (Chou and Shen, 2007), and PrediSi (Hiller et al., 2004). To further assess protein location prediction, researchers are encouraged to look for β-barrel proteins and dismiss proteins containing transmembrane α-helix (beware that this conformational ‘rule’ does not apply to lipoproteins, only outer membrane proteins).
There are several techniques to assess experimentally the location of predicted proteins and they vary according to the bacterium being studied. Cell fractionation followed by Western blot, indirect immunofluorescence (unfixed samples), and cell surface proteolysis among other techniques may be suitable as well. To assess the localization of candidate proteins of the meningococcus, we produced serum against candidate proteins, probed intact bacteria with this serum, and added FITC-conjugated secondary antibodies and analysed single-cells by flow cytometry. The detection of fluorescence would confirm the surface exposure of a given candidate protein as demonstrated in Figure 2. Please refer to Newcombe et al., 2014 for more details on this technique and other techniques we used.


Figure 2. Representative data of flow cytometry experiment to assess whether a  candidate protein is surface exposed or not. Cells probed with sera against surface exposed proteins and FITC show a stronger signal (grey line) than the negative control  (black line). Reproduced from Newcombe et al. (2014).

Notes

  1. Serum samples should be selected based on immunity against the bacteria of interest, this can be healthy sera which has shown to be confer immunity or sera specifically raised against the target organism. The antibody titre against the antigen will determine the amount of serum required to immunoprecipitate sufficient antigenic proteins.
  2. In addition to SDS-PAGE of immunoprecipitated proteins, we also recommend performing Western blots to confirm the presence of immunogenic proteins. The presence of other protein bands would indicate the extraction of non-specific proteins. It is essential to run controls including an antibody negative sample. It will be necessary to ensure the serum contains antibodies to the bacteria. The titre of the serum will affect the amount required to immunoprecipitate detectable levels of specific immunogenic proteins. The use of serum against non-surface proteins (e.g., periplasmic proteins, inner membrane proteins, etc.) is also advised to ensure there are no contamination of proteins that are not surface exposed.
  3. Step A7 of immunoprecipitation can be done using the spin columns or carefully pipetting off the supernatant.
  4. Mass spectrometry parameters are specific to the instrumentation available to the researchers. We report here specifications that we used, but researchers should refer to their own equipment instructions to find out how to better adjust their equipment. There are also other ways to analyse MS files (Procedure D). Other software and methodologies are available. We demonstrate here what software we incorporated in our analysis. It is recommended that the immunoprecipitated sample be split and or multiple samples analysed to enhance the reliability of the data.
  5. The preparation of peptides for the mass spectrometer analysis, the mass spectrometer data collection, and subsequent data analysis will depend on the facility performing the analysis. If this is in house or to be outsourced discuss the sample preparation before preparing the samples. All facilities have set procedures to ensure maximum output.

Recipes

  1. CAB medium
    39 g Columbia agar base
    Distilled water to 1 L
    Autoclave (121 °C, 15 min)
    Cool to 50 °C and add 6% sterile defibrinated blood
  2. PBS plus 1% BSA
    1 g BSA
    PBS to 100 ml final volume, keep refrigerated
  3. Solubilization buffer
    3.0028 g Tris, 50 mM, pH 7.8
    4.38 g NaCl, 150 mM
    0.146 g EDTA (or dilute from 500 mM stock solution), 1 mM
    1% (v/v) Triton X-100
    1 g sodium deoxycholate, 0.2%
    0.5 g SDS (or dilute from 10% stock solution–weight in fume hood), 0.1%
    Distilled water (to a final volume of 500 ml)
  4. 1% SDS
    1 g SDS
    100 ml LC/MS water
    Weight in fume hood
  5. 1 M dithiothreitol stock solution
    1.542 g DTT
    LC/MS water to 10 ml, keep at -20 °C
    Weight in fume hood
  6. 1 M Iodoacetamide stock solution
    1.84 g IAA
    Distilled LC/MS water to 10 ml
  7. 25 mM ammonium bicarbonate
    0.998 g ammonium bicarbonate
    LC/MS water to 500 ml
  8. Acetonitrile (50% v/v) in 25 mM ammonium bicarbonate
    50 ml acetonitrile
    50 ml ammonium bicarbonate (see above)
  9. Acetonitrile (50% v/v)
    50 ml acetonitrile
    50 ml LC/MS water
  10. Solvent A for HPLC
    2 ml acetonitrile
    0.1 ml formic acid
    97.9 ml LC/MS water
  11. Solvent B for HPLC
    90 ml acetonitrile
    0.1 ml formic acid
    9.9 ml LC/MS water
  12. 0.1% formic acid/50% acetonitrile solution
    0.1 ml formic acid
    50 ml acetonitrile
    49.9 ml LC/MS water

Acknowledgments

The authors declare no conflicts of interest. This protocol has been previously described by us (Newcombe et al., 2014). Sanofi Pasteur funded this study. CNPq and CAPES funded the adaptation to other Gram-negative species.

References

  1. Chou, K. C. and Shen, H. B. (2007). Signal-CF: a subsite-coupled and window-fusing approach for predicting signal peptides. Biochem Biophys Res Commun 357(3): 633-640.
  2. Hiller, K., Grote, A., Scheer, M., Munch, R. and Jahn, D. (2004). PrediSi: prediction of signal peptides and their cleavage positions. Nucleic Acids Res 32(Web Server issue): W375-379.
  3. Juncker, A. S., Willenbrock, H., Von Heijne, G., Brunak, S., Nielsen, H. and Krogh, A. (2003). Prediction of lipoprotein signal peptides in Gram-negative bacteria. Protein Sci 12(8): 1652-1662.
  4. Mendum, T. A., Newcombe, J., McNeilly, C. L. and McFadden, J. (2009). Towards the immunoproteome of Neisseria meningitidis. PLoS One 4(6): e5940.
  5. Newcombe, J., Mendum, T. A., Ren, C. P. and McFadden, J. (2014). Identification of the immunoproteome of the meningococcus by cell surface immunoprecipitation and MS. Microbiology 160(Pt 2): 429-438.
  6. Petersen, T. N., Brunak, S., von Heijne, G. and Nielsen, H. (2011). SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods 8(10): 785-786.
  7. Shen, H. B. and Chou, K. C. (2010). Gneg-mPLoc: a top-down strategy to enhance the quality of predicting subcellular localization of Gram-negative bacterial proteins. J Theor Biol 264(2): 326-333.
  8. Vogel, U., Taha, M. K., Vazquez, J. A., Findlow, J., Claus, H., Stefanelli, P., Caugant, D. A., Kriz, P., Abad, R., Bambini, S., Carannante, A., Deghmane, A. E., Fazio, C., Frosch, M., Frosi, G., Gilchrist, S., Giuliani, M. M., Hong, E., Ledroit, M., Lovaglio, P. G., Lucidarme, J., Musilek, M., Muzzi, A., Oksnes, J., Rigat, F., Orlandi, L., Stella, M., Thompson, D., Pizza, M., Rappuoli, R., Serruto, D., Comanducci, M., Boccadifuoco, G., Donnelly, J. J., Medini, D. and Borrow, R. (2013). Predicted strain coverage of a meningococcal multicomponent vaccine (4CMenB) in Europe: a qualitative and quantitative assessment. Lancet Infect Dis 13(5): 416-425.
  9. Wheeler, J. X., Vipond, C. and Feavers, I. M. (2007). Exploring the proteome of meningococcal outer membrane vesicle vaccines. Proteomics Clin Appl 1(9): 1198-1210.
  10. Yu, C. S., Chen, Y. C., Lu, C. H. and Hwang, J. K. (2006). Prediction of protein subcellular localization. Proteins 64(3): 643-651.
  11. Yu, C. S., Lin, C. J. and Hwang, J. K. (2004). Predicting subcellular localization of proteins for Gram-negative bacteria by support vector machines based on n-peptide compositions. Protein Sci 13(5): 1402-1406.
  12. Yu, N. Y., Wagner, J. R., Laird, M. R., Melli, G., Rey, S., Lo, R., Dao, P., Sahinalp, S. C., Ester, M., Foster, L. J. and Brinkman, F. S. (2010). PSORTb 3.0: improved protein subcellular localization prediction with refined localization subcategories and predictive capabilities for all prokaryotes. Bioinformatics 26(13): 1608-1615.

简介

脑膜炎球菌(脑膜炎奈瑟氏球菌)仍然是全球人类健康的重大威胁。这种革兰氏阴性细菌导致感染个体的残疾和死亡率升高。尽管有几种可用的疫苗,目前还没有针对所有循环脑膜炎球菌菌株的通用疫苗(Vogel等人,2013)。在这里,我们描述了一种能够识别仅在细胞表面暴露的蛋白质在免疫中发挥作用的新方案,为该研究领域提供了一种更直接的方法来鉴定新的疫苗靶标。即使使用脑膜炎奈瑟氏球菌作为本文所述方案中的模型,该方案可用于任何革兰氏阴性细菌,提供修饰和优化以使其适应不同的细菌和疾病特征(例如薄膜脆性,生长方法,血清抗体水平,等等)。


背景 尝试开发针对N型的新型疫苗。脑膜炎脑膜炎常常依赖于2D SDS-PAGE(二维十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳)和蛋白质印迹,随后MS(质谱)(Wheeler等人,2007))。然而,这种方法采用全细胞裂解物,鉴定出不具有疫苗潜力的大量蛋白质(Mendum等人,2009)。因此,我们旨在开发一种能够鉴别可能在免疫中起重要作用的细胞表面暴露蛋白质的方法。简言之,我们的方案包括生长感兴趣的病原体,用免疫个体的血清免疫沉淀表面抗原,并通过液相色谱 - 串联质谱鉴定免疫沉淀的蛋白质。我们能够使用这种新方案鉴定23种脑膜炎球菌表面抗原,其中一些是市售疫苗的组成部分(Newcombe等人,2014)。我们也已经将此协议适用于其他革兰氏阴性细菌,并获得了有希望的结果:我们确定了以前描述的表面暴露蛋白质,其中许多蛋白质已被测试为疫苗或诊断测试候选者。这些结果表明,这是一种强大的技术,可以应用于各种革兰氏阴性细菌,并且能够产生高质量的结果,可以通过无数应用(例如,疫苗)进一步利用,诊断等。

关键字:革兰氏阴性, 免疫蛋白质组, 免疫沉淀, 细胞表面抗原, 外膜蛋白, 暴露的抗原

材料和试剂

  1. 一次性培养皿(克伦威尔集团,目录号:STS3855002B)
  2. L型细胞扩散器(Fisher Scientific,目录号:14-665-231)
  3. 一次性接种环(Sigma-Aldrich,目录号:I8388)
  4. 1.5ml微量离心管(Corning,Axygen ,目录号:MCT-150-C)
  5. 蛋白质LoBind管(Eppendorf,目录号:022431102)
  6. 20 ml塑料普通(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:128BFS)
  7. 塑料密封袋(Fisher Scientific,目录号:10366984)
    制造商:MINIGRIP,目录号:BAJ-340-091N。
  8. Pierce TM旋转柱(Thermo Fisher Scientific,Thermo Scientific TM,目录号:69725)
  9. 一次性无菌手术刀10号(WMS,目录号:W259)
  10. 10μl过滤嘴(STARLAB INTERNATIONAL,TipOne ®,目录号:S1121-3810)
  11. 20μl过滤嘴(STARLAB INTERNATIONAL,TipOne ®,目录号:S1120-1810)
  12. 200μl过滤嘴(STARLAB INTERNATIONAL,TipOne ®,目录号:S1120-8810)
  13. 1,000μl过滤嘴(STARLAB INTERNATIONAL,TipOne ®,目录号:S1122-1830)
  14. 脑膜炎奈瑟氏球菌(L9153和MC58,英国公共卫生局)
  15. 磷酸盐缓冲盐水(PBS)(Thermo Fisher Scientific,Gibco TM,目录号:10010001)
  16. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A9418)
  17. 人血清(Sigma-Aldrich,目录号:H6914)
  18. 疾病状态血清(获得这种变化,取决于正在研究的病原体)
  19. Pierce TM 蛋白A/G UltraLink TM树脂(Thermo Fisher Scientific,Thermo Scientific TM,目录号:53132)
  20. SeeBlue ®蛋白质标记(Thermo Fisher Scientific,Novex TM,目录号:LC5925)
  21. 二硫苏糖醇(DTT)(Sigma-Aldrich,目录号:43815)
  22. 碘乙酰胺(Sigma-Aldrich,目录号:I6125)
  23. (Thermo Fisher Scientific,Novex< sup>,目录号:NP0004)的10x样品还原剂)
  24. Invitrogen(株)Supex TM Supex TM Supex TM Supex TM 4/LDS加载缓冲液(Thermo Fisher Scientific,Novex TM,目录号:NP0007 )
  25. Invitrogen(株)Supp。TM Supex TM Supp。TM Supe12%Bis-Tris 1 mm-10孔(Thermo Fisher Scientific,Invitrogen TM >,目录号:NP0341BOX)
  26. Invitrogen Supreme TM NuPAGE TM MOPS运行缓冲液20x(Thermo Fisher Scientific,Novex TM,目录号:NP000102
  27. SimplyBlue TM 安全污渍(Thermo Fisher Scientific,Novex TM ,目录号:LC6060)
  28. 用于HPLC-MS的乙腈(Fisher Scientific,目录号:10616653)
  29. 碳酸氢铵(Sigma-Aldrich,目录号:09830)
  30. 胰蛋白酶金,质谱级(Promega,目录号:V5280)
  31. 哥伦比亚血琼脂碱(Oxoid,目录号:CM0331)
  32. 马血,脱纤维(Oxoid,目录号:SR0050)
  33. Tris碱(Roche Diagnostics,目录号:10708976001)
  34. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S9625)
  35. EDTA(Sigma-Aldrich,目录号:E6758)
  36. Triton X-100(Sigma-Aldrich,目录号:X100)
  37. 脱氧胆酸钠(Sigma-Aldrich,目录号:30970)
  38. 十二烷基硫酸钠(SDS)(Sigma-Aldrich,目录号:L3771)
  39. 甲酸LC/MS(Fisher Scientific,目录号:A117-50)
  40. 用于HPLC-MS的水(Fisher Scientific,目录号:10777404)
  41. 甲醇(Sigma-Aldrich,目录号:179957-1L)
  42. 乙酸(Sigma-Aldrich,目录号:A6283)
  43. CAB培养基(见食谱)
  44. PBS加1%BSA(参见食谱)
  45. 溶解缓冲液(见配方)
  46. 1%SDS(参见食谱)
  47. 1 M二硫苏糖醇储液(参见食谱)
  48. 1 M碘乙酰胺(见配方)
  49. 25 mM碳酸氢铵(见配方)
  50. 25%碳酸氢铵中的乙腈(50%v/v)(见配方)
  51. 乙腈(50%v/v)(见配方)
  52. 溶剂A用于HPLC(参见食谱)
  53. HPLC溶剂B(见配方)
  54. 0.1%甲酸/50%乙腈溶液(参见食谱)

设备

  1. Gilson Pipette Pipetman Classic P2(Gilson,目录号:F144801)
  2. Gilson Pipette Pipetman Classic P20(Gilson,目录号:F123600)
  3. Gilson Pipette Pipetman Classic P200(Gilson,目录号:F123601)
  4. Gilson Pipette Pipetman经典P1000(Gilson,目录号:F123602)
  5. Eppendorf ®微型离心机5415R(Eppendorf,型号:5415 R)
  6. BOD低温冷藏培养箱(VWR,型号:BOD Incubator 2005,目录号:35960-056)
  7. CO 2培养箱(LEEC,型号:Culture Safe Precision 190D)
  8. 斯图尔特旋转摇杆(Cole-Parmer,Stuart,型号:SSL3)
  9. Vortex Mixer SA8(Cole-Parmer,Stuart,型号:SA8)
  10. 超声波浴(Fisher Scientific,型号:Fisherbrand TM S系列,目录号:10611983)
  11. Ultimate 3000纳米HPLC系统(Thermo Fisher Scientific,Thermo Scientific TM,型号:UltiMate TM 3000/RSL Cnano系统)
  12. Acclaim TM C-18 Pepmap TM 列(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:164569)
  13. QSTAR ® XL混合LC/MS/MS系统(Applied Biosystems,型号:QSTAR XL Hybrid LC/MS/MS系统)
  14. Eppendorf MixMate TM (Fisher Scientific,目录号:21-379-00)
    制造:Eppendorf,目录号:022674200。
  15. Eppendorf ThermoStat Plus可互换块加热器(Eppendorf,型号:ThermoStat Plus,目录号:022670204)
  16. Eppendorf Vacufuge TM 浓缩器(Eppendorf,型号:Vacufuge TM浓缩器,目录号:022820001)
  17. Heraeus管辊Spiramix(Fisher Scientific,目录号:10600653)
    制造商:Thermo Fisher Scientific,Thermo ScientificTM,产品编号:DS507。
  18. 通风柜

软件

  1. Chromeleon v6.8或更高版本(Dionex,Thermo Fisher Scientific)
  2. 分析师QS 1.1(Applied Biosystems)
  3. Mascot v2.2.04或更高版本(Matrix Science,London)

程序

  1. 细胞表面免疫沉淀
    1. 成长。来自冷冻原料的脑膜炎脑膜炎:用无菌200μl提取物获得少量冷冻培养物,接种哥伦比亚琼脂碱(CAB)板并铺展以产生单个菌落。在35℃下用5%CO 2孵育过夜(〜18小时)。收集20-30个菌落,并使用无菌环扩散到CAB板的整个表面,并在35℃下用5%CO 2孵育4小时。个别殖民地在这个阶段不会被看到。
    2. 通过向板中加入PBS并使用无菌环将菌落混入PBS中来回收细菌。使用P1000微量移液管取出PBS /细菌悬浮液(每片1ml,每次免疫沉淀使用半片),室温下以13,000 xg离心2分钟,用1体积PBS洗涤沉淀3次加1%BSA。对于这些和随后的洗涤步骤,通过在室温下以13,000 x g离心2分钟,沉淀细胞,除去上清液,加入新鲜的缓冲液并重悬浮颗粒。
      注意:
      1. 如果使用液体培养而不是平板,我们建议在对数生长期和在有利于表达毒力因子的条件下收获细胞。我们建议研究人员利用其生物体进行生长曲线,或者了解生物体如何在液体培养基中生长以便能够正确识别生长期。
      2. 离心条件可能根据所讨论的细菌而变化。裂解细胞的存在将增加对非表面蛋白质的选择。
      3. 尽一切努力确保早期离心步骤不会破坏细胞,同时去除分泌的蛋白质或裂解的死细胞的组分(见注2)。
    3. 在1ml血清样品中悬浮沉淀(对照或试验[分别为材料和试剂列表的项目17和18]),并在4℃下旋转90分钟。
      注意:在对照血清样品中鉴定的蛋白质在将来的分析中应该被忽略,因为它们可能在针对病原体的免疫应答中起不到任何作用。研究人员应确保他们所控制的血清对他们正在调查的病原体确实是阴性的。
    4. 将细胞(13,000 x g,5分钟,RT)造粒,并用PBS加1%BSA洗涤两次。
    5. 将细胞重悬于1ml溶解缓冲液中,37℃孵育60分钟
    6. 离心样品(13,000 x g,60分钟,RT),收集上清液,并用蛋白A/G UltraLink珠(50μl)在4℃下搅拌或滚动孵育18小时。
    7. 离心样品(2,500 x g,2分钟,RT),收集珠子。
    8. 用1体积溶解缓冲液洗涤珠子5次(2,500×g,2分钟)。
    9. 洗脱抗体:1体积SDS(1%)和沸腾(10分钟)的抗原复合物
    10. 加入4体积的水并涡旋洗脱蛋白质。离心机(2,500 x g,3 min,RT),收集上清液;体积的增加允许在涡旋期间更有效地去除蛋白质。
    11. 将样品浓缩至50μl,使SDS浓度回复至1%(使用Speed-Vac在45°C约45分钟)。此时,可以将样品与加载缓冲液混合并在SDS PAGE凝胶上运行。继续步骤B1。如果样品正在准备进行质谱分析,则蛋白质可以在分离前进行还原和烷基化。通过加入DTT至终浓度为10 mM并煮沸2分钟(50μl,加入0.5μl1 M DTT原液),减少洗脱的蛋白质。
    12. 加入碘乙酰胺至终浓度为50 mM(50μl,加入2.5μl1M IAA储备溶液),并在黑暗中室温孵育30分钟。

  2. 1D SDS-PAGE和样品消化
    1. 将减少的烷基化样品与加载缓冲液(材料和试剂列表的项目23)混合并煮沸5分钟。
    2. 通过SDS-PAGE(12%,1 mm厚,10 cm长)分离蛋白质。
    3. 用SimplyBlue TM染色1小时,用超纯水去污(根据制造商的说明书,见图1)。


      图1.脑膜炎球菌的免疫沉淀(IP)表面蛋白的代表性图像。 转载自Newcombe 等人(2014)。

    4. 根据样品蛋白质浓度,根据需要切割凝胶泳道,并将切片置于低蛋白质结合管中 注意:一个完整的凝胶泳道可以切割成20个切片,但是只需要运行一个缩短的凝胶并将其切成5个切片。
    5. 用50%乙腈在25mM碳酸氢铵中洗涤凝胶片两次,在室温下涡旋15分钟以除去污渍用量足够的体积以覆盖凝胶片。
    6. 通过与100%乙腈(20分钟,室温)孵育脱水凝胶切片
    7. 使用Speed-Vac干燥(约20分钟)的干凝胶片。
    8. 在含有修饰胰蛋白酶的25mM碳酸氢铵(12.5ng /μl,终体积将取决于凝胶切片的大小,4℃10分钟)中再水化。如果需要,加入更多的25mM碳酸氢铵以覆盖凝胶片,在37℃下孵育过夜
    9. 除去消化液,通过与30μl0.1%甲酸/50%乙腈孵育20-30分钟提取消化的蛋白质20分钟,振荡,然后在超声波浴(40 kHz)中重复一次,每次收集上清液。 />
    10. 使用Speed-Vac彻底干燥提取的消化蛋白,并重悬于20μl水中。储存于-20°C。

  3. 液相色谱 - 电喷雾离子化MS/MS(请参见注释)
    1. 使用Ultimate 3000 HPLC和C18 pepman(Dionex)柱在40℃,350nl/min,2-50%溶剂B梯度洗脱30分钟,90%溶剂B,用Chromeleon软件控制5分钟,分离肽。 />
    2. 将QSTAR XL设置为2.3 kV电喷雾,8个任意单位的帘式气体,以及400至1200 m/z的1秒测量,充电状态2-4。
    3. 通过分析员QS 1.1软件获取信息数据采集的MS数据。
    4. 为MS/MS选择最大强度的产品离子,其中m/z 为65和1,200 amu。

  4. 数据库分析(请参见注释)
    1. 向Mascot Server(v2.2.04; Matrix Science)提交MS/MS谱的峰列表,并使用MS/MS离子搜索程序进行分析。
    2. 允许多达一个缺失的胰蛋白酶切割与固定修饰的氨基甲基(C)和可变的氧化修饰(M)。
    3. 将肽和MS/MS片段的耐受度分别设置为1.2和0.6Da,并选择MH 2 + 和MH 3+/sup>作为前体充电状态
    4. 搜索确定的蛋白质与国家生物技术信息中心非冗余(NCBInr)数据库,其分类集为。脑膜炎病毒分类法(分类代码487)。使用正在使用的生物体的适当数据库分析数据。

数据分析

一旦获得了确定的蛋白质的列表,应检查它们以确保所有的蛋白质都被表面暴露。这可以使用诸如PSORTb v3.0(Yu等人,2010),CELLO v2.5(Yu et al。,2004)和2006)或Gneg-mPLoc(Shen and Chou,2010)。搜索信号肽或脂质化也是有用的。我们推荐使用以下工具:LipoP 1.0(Juncker,2003),SignalP 4.1(Petersen等人,2011),Signal-CF(Chou and Shen,2007)和PrediSi(Hiller < et al。,2004)。为了进一步评估蛋白质位置预测,鼓励研究人员寻找β-桶蛋白,并排除含有跨膜α-螺旋的蛋白质(请注意,此构象"规则"不适用于脂蛋白,仅适用于外膜蛋白)。
有几种技术来实验地评估预测的蛋白质的位置,并且它们根据正在研究的细菌而变化。细胞分级随后是Western印迹,间接免疫荧光(未固定的样品)和细胞表面蛋白水解等技术也是合适的。为了评估脑膜炎球菌候选蛋白的定位,我们制备了候选蛋白的血清,用该血清探测完整细菌,并加入FITC缀合的二抗,并通过流式细胞术分析单细胞。如图2所示,荧光的检测将证实给定候选蛋白的表面暴露。请参阅2014年的Newcombe等人。有关此技术和我们使用的其他技术的更多细节。 />

图2.流式细胞术实验的代表性数据,以评估候选蛋白是否是表面暴露的。用表面暴露蛋白和FITC的血清探测的细胞显示出更强的信号(灰线)阴性对照(黑线)。从Newcombe等人转载(2014)。

笔记

  1. 应根据对感兴趣的细菌的免疫力来选择血清样品,这可以是已显示赋予免疫力的健康血清或针对目标生物体特异提高的血清。针对抗原的抗体滴度将决定免疫沉淀足够的抗原蛋白所需的血清量
  2. 除免疫沉淀蛋白质的SDS-PAGE之外,我们还建议进行蛋白质印迹以确认免疫原性蛋白质的存在。其他蛋白质条带的存在将表明非特异性蛋白质的提取。运行对照包括抗体阴性样本是至关重要的。有必要确保血清含有对细菌的抗体。血清的滴度将影响免疫沉淀可检测水平的特异性免疫原性蛋白质所需的量。还建议使用血清对非表面蛋白质(例如,周质蛋白质,内膜蛋白质等)进行检测,以确保不会污染不属于蛋白质的蛋白质表面暴露。
  3. 免疫沉淀的步骤A7可以使用旋转柱或小心地从上清液中移出来进行
  4. 质谱参数特异于研究人员可用的仪器。我们在这里报告我们使用的规格,但研究人员应参考自己的设备说明,了解如何更好地调整设备。还有其他方式来分析MS文件(过程D)。其他软件和方法可用。我们在这里展示了我们在分析中纳入的软件。建议将免疫沉淀的样品分开或分析多个样品,以提高数据的可靠性。
  5. 用于质谱仪分析,质谱仪数据收集和随后数据分析的肽的制备将取决于进行分析的设备。如果这是在内部或外包在准备样品前讨论样品制备。所有设施都设定了确保最大输出的程序。

食谱

  1. CAB媒体
    39克哥伦比亚琼脂基地
    蒸馏水至1升
    高压灭菌器(121℃,15分钟)
    冷却至50°C,加入6%无菌去纤维化血液
  2. PBS加1%BSA
    1克BSA
    PBS至100ml最终体积,保持冷藏
  3. 溶解缓冲液
    3.0028g Tris,50mM,pH7.8。
    4.38克NaCl,150毫升 0.146g EDTA(或从500mM储备溶液稀释),1mM
    1%(v/v)Triton X-100
    1g脱氧胆酸钠,0.2%
    0.5克SDS(或从通风橱中的10%储液重量稀释),0.1%
    蒸馏水(最终体积为500毫升)
  4. 1%SDS
    1克SDS
    100 ml LC/MS水 通风柜重量
  5. 1 M二硫苏糖醇溶液
    1.542克DTT
    LC/MS水至10ml,保持-20°C 通风柜重量
  6. 1 M碘乙酰胺储备溶液
    1.84 g IAA
    蒸馏LC/MS水至10 ml
  7. 25 mM碳酸氢铵
    0.998克碳酸氢铵
    LC/MS水至500 ml
  8. 乙腈(50%v/v)在25 mM碳酸氢铵中 50ml乙腈
    50ml碳酸氢铵(见上文)
  9. 乙腈(50%v/v)
    50ml乙腈
    50ml LC/MS水
  10. 溶剂A用于HPLC
    2ml乙腈
    0.1毫升甲酸
    97.9 ml LC/MS水
  11. 溶剂B用于HPLC
    90 ml乙腈 0.1毫升甲酸
    9.9 ml LC/MS水
  12. 0.1%甲酸/50%乙腈溶液
    0.1毫升甲酸
    50ml乙腈
    49.9 ml LC/MS水

致谢

作者声明没有利益冲突。此协议之前已经被我们描述过(Newcombe等人,,2014)。赛诺菲巴斯德资助这项研究。 CNPq和CAPES资助了其他革兰氏阴性物种的适应性。

参考

  1. Chou,KC and Shen,HB(2007)。  信号-CF:用于预测信号肽的亚位点耦合和窗口融合方法。 Biochem Biophys Res Commun 357(3):633-640。
  2. Hiller,K.,Grote,A.,Scheer,M.,Munch,R.and Jahn,D。(2004)。 PrediSi:信号肽及其切割位置的预测。核酸Res 32(Web Server issue):W375- 379.
  3. Juncker,AS,Willenbrock,H.,Von Heijne,G.,Brunak,S.,Nielsen,H.and Krogh,A。(2003)。  革兰氏阴性菌中脂蛋白信号肽的预测蛋白质科学12(8): 1652-1662。
  4. Mendum,TA,Newcombe,J.,McNeilly,CL和McFadden,J。(2009)。  走向脑膜炎奈瑟氏球菌免疫蛋白质。 PLoS One 4(6):e5940。
  5. Newcombe,J.,Mendum,TA,Ren,CP and McFadden,J。(2014)。通过细胞表面免疫沉淀和MS鉴定脑膜炎球菌的免疫蛋白体。 160(Pt 2):429-438。
  6. Petersen,TN,Brunak,S.,von Heijne,G.和Nielsen,H。(2011)。 SignalP 4.0:区分来自跨膜区域的信号肽。 Nat方法8(10):785-786。
  7. Shen,HB and Chou,KC(2010)。  Gneg -mPLoc:自上而下的策略,以提高预测革兰氏阴性细菌蛋白质亚细胞定位的质量。 J Theor Biol 264(2):326-333。
  8. Vogel,U.,Taha,MK,Vazquez,JA,Findlow,J.,Claus,H.,Stefanelli,P.,Caugant,DA,Kriz,P.,Abad,R.,Bambini,S.,Carannante,A ,Deghmane,AE,Fazio,C.,Frosch,M.,Frosi,G.,Gilchrist,S.,Giuliani,MM,Hong,E.,Ledroit,M.,Lovaglio,PG,Lucidarme,J.,Musilek ,M.,Muzzi,A.,Oksnes,J.,Rigat,F.,Orlandi,L.,Stella,M.,Thompson,D.,Pizza,M.,Rappuoli,R.,Serruto,D.,Comanducci ,M.,Boccadifuoco,G.,Donnelly,JJ,Medini,D.and Borrow,R。(2013)。  欧洲脑膜炎球菌多组分疫苗(4CMenB)的预测应变覆盖率:定性和定量评估。柳叶刀感染D >是 13(5):416-425。
  9. Wheeler,J.X。,Vipond,C.and Feavers,I.M。(2007)。 探索脑膜炎球菌外膜囊泡疫苗的蛋白质组。 蛋白质组学临床应用 1(9):1198-1210。
  10. Yu,CS,Chen,YC,Lu,CH和Hwang,JK(2006)。  蛋白质亚细胞定位的预测。 64(3):643-651。
  11. Yu,CS,Lin,CJ和Hwang,JK(2004)。通过基于n肽组合物的支持载体机器预测革兰氏阴性细菌蛋白质的亚细胞定位。蛋白质Sci 13(5):1402-1406。
  12. Yu,NY,Wagner,JR,Laird,MR,Melli,G.,Rey,S.,Lo,R.,Dao,P.,Sahinalp,SC,Ester,M.,Foster,LJ and Brinkman,FS(2010 )。 PSORTb 3.0:改进的蛋白质亚细胞定位预测本地化子类别和所有原核生物的预测能力。生物信息学 26(13):1608-1615。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:da Cunha, C. P., Newcombe, J., Dellagostin, O. A. and McFadden, J. (2017). Immunoprecipitation of Cell Surface Proteins from Gram-negative Bacteria. Bio-protocol 7(9): e2250. DOI: 10.21769/BioProtoc.2250.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。