搜索

Purification and TEM of Afp and Its Variants
据食原噬菌体及其变体的纯化和透射电子显微分析   

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 Serratia entomophila antifeeding prophage (Afp), forms a phage-tail-like particle (tailocin) that causes cessation of feeding activity of the New Zealand grass grub, Costelytra zealandica. Here, we describe the more detailed purification protocol for Afp particles and its variants which is based on the procedure described in our original publication (Rybakova et al., 2013). The purification procedure includes inducing Escherichia coli (E. coli) cells harbouring afp genes under arabinose-inducible promoter for 24 h. The cells are harvested and sonicated on ice followed by DNAse treatment and centrifugation. The supernatant is then filter sterilised and applied to the size exclusion chromotography (SEC) column. The fractions containing Afp or its variants are pooled and ultracentrifuged. The supernatant is removed and the transparent pellet is resuspended in the residual buffer. The procedure results in Afp particles or variants thereof that are approximately 70% pure. The Afp particles are negatively stained and visualized using Transmission electron microscopy (TEM).

Materials and Reagents

Note: All solutions, unless otherwise specified, were prepared with distilled deionised Millipore-microfiltered (MilliQ) water, autoclaved for 15 min at 1.1 kg/cm2 at 121 °C and stored at room temperature. If antibiotics or supplements were to be added, the medium was cooled to a temperature of 50-60 °C before addition of antibiotic or supplement solution. The following antibiotics were used at stated concentrations (μg/ml): ampicillin, 100; chloramphenicol, 30; and spectinomycin, 100.

  1. E. coli strains containing afp genes under arabinose promoter
    Note: Please refer to the original manuscript (Rybakova et al., 2013) for strains and antibiotic resistance description.
  2. LB-agar (Merck KGaA, catalog number: VM459383236 )
  3. DNase (Roche Diagnostics, catalog number: 04716728001 )
  4. 0.7 % filtered (pore size 0.2 um) uranyl acetate diluted in sterile water (pH 5.0) (Electron Microscopy Sciences, catalog number: 22400 )
  5. Sephacryl S-400 HR resin (GE, http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/en/GELifeSciences/products/AlternativeProductStructure_17395/17060901)
  6. 20% L-Arabinose (Acros organics) (Thermo Fisher Scientific, catalog number: AC104985000 ) (see Recipes)
  7. 0.4x Luria-Bertani broth (LB) (Life Technologies, catalog number: 12795-027 ) (see Recipes)
  8. 0. 2% sodium azide (Sigma-Aldrich, catalog number: S8032 ) (see Recipes)
  9. 250 mM TBS buffer (see Recipes)
  10. TM buffer (see Recipes)

Equipment

  1. Spectrophotometer
  2. Orbital mixer incubator (Raytek Corporation)
  3. 1.7 ml microcentrifuge tubes
  4. Eppendorf centrifuges
  5. Sanyo soniprep 150 sonicator (18 Ω) (Panasonic Corporation)
  6. 0.45-µm-pore-size cellulose acetate filters (Advantec MFS, catalog number: 03CP045AN )
  7. 50 ml BD Falcon Tubes
  8. Sephacryl S-400 HR beads (GE Healthcare, catalog number: 17-0609-10 ) for size exclusion chromatography (SEC)
  9. The SEC column with a bed volume of 1.5 by 46 cm (Bio-Rad Laboratories, model: 737-4155 )
  10. 1.5 x 50 cm (D x L) column (Farmacia)
  11. BioLogic LP system (Bio-Rad Laboratories, model: 731-8350 )
  12. Beckman coulter Optima TM L-100K ultracentrifuge (Beckman Coulter, model: 392050 )
  13. Polaron E5100 SEM coating unit
    Note: This unit is from before 2000 and is no longer available, however a similar unit that is equivalent can be found at http://www.quorumtech.com/products/glow-discharge-system-free-standing.html.
  14. Whatman #1 filter paper (Whatman)
  15. Plastic-coated 200-mesh grid (ProSciTech, catalog number: GCU200 )
  16. Morgagni 268D TEM (FEI)
  17. Megapixel III digital camera
    Note: The catalog numbers and model numbers are not important for reproducibility.
  18. Polypropylen tubes used for the size exclusion chromotography (polypropylene 12 x 75 mm, 5 ml) (Labserv, catalog number: LBS504N )
  19. Polypropylen tubes for ultracentrifugation (polycarbonate centrifuge bottles with cap assembly 16 x 76 mm, capacity 10.4 ml) (Beckman Coulter, catalog number: 355603 )
  20. Falcon tubes (Thermo Fisher Scientific, catalog number: 14-432-22 )
  21. Protect microorganism preservation system (Technical Service Consultants) for preservation of E. coli cells at -80 °C
    Note: When required, a single inoculated bead was removed from the vial and dropped into a LB broth containing the appropriate antibiotic.

Procedure

Note: Centrifugation steps were performed at 4 °C unless otherwise noted.

  1. Afp purification protocol
    1. A single inoculating bead (Protect Microorganism Preservation System) from the -80 °C stock of an appropriate Afp or its derivatives bearing E. coli strain is used to inoculate 3 ml of LB with the respective antibiotics.
    2. The culture is incubated overnight at 37 °C with agitation at 250 rpm (overnight culture).
    3. 1 ml of the overnight culture is used to inoculate 50 ml of fresh LB with appropriate antibiotic/s.
    4. The 50 ml of the cell culture is grown at 37 °C with agitation to an OD600 of 1.0.
      Note: OD600 measurements are taken from the cell cultures diluted with the LB media at ratios of 1:10. A cuvette with LB medium is used as a blank.
    5. The cells are transferred into sterile falcon tubes (50 ml) and harvested by centrifugation at 2,268 x g for 10 min at room temperature.
    6. The cell pellets are resuspended in fresh 50 ml 0.4x LB broth with respective antibiotic/s.
      Note: Initially 1 ml of the 0.4x LB broth is added to each pellet and the pellet is resuspended by vigorous taping on the tube. Then the tube is filled up to 50 ml with 0.4x LB broth and the content of the tube is transferred to the same 50 ml flask as the culture was grown. Antibiotics are added directly to the flask.
    7. 500 μl of steril filtered 20% arabinose (final concentration 0.2%) is added to each 50 ml culture to induce the protein expression.
      Note: After arabinose is added, avoid shaking the cultures.
    8. The cultures are incubated for a further 24 h at 22 °C at 40 rpm.
    9. The induced cells are harvested by centrifugation for 10 min at 2,268 x g.
      Note: Higher centrifugation speed (4,629 x g for 10 min) may be nessessary if pellet is too loose.
    10. The pellet from each 50 ml culture is re-suspended at 4 °C in 0.8 ml pre-chilled TM buffer.
    11. The resultant approximately 0.9 ml re-suspensions are transferred into 1.7 ml microcentrifuge tubes and subjected to three 20-sec rounds of sonication using a Sanyo Soniprep 150 Sonicator (18 Ω). At least 1 min of incubation on ice is included between sonication steps.
    12. One μl (10 units) of the DNase is added and the sample is left at room temperature for 10 min.
    13. Following the DNase incubation step the suspension is centrifuged at 16,000 x g for 10 min at 4 °C.
    14. The pellets are discarded and clear supernatants containing soluble recombinant protein are filtered through 0.45-µm-pore-size filters.
      Notes:
      1. If the samples need to be stored for 1-2 days, 0.02% (final concentration) NaN3 is added to each tube.
      2. Storage longer than 2 days was not tested; however for a better reproducibility it is recomended to proceed with the purification immediately after this step.
    15. The resin is packed into a 1.5 x 50 cm (D x L) column according to the manufacturers’ instructions, resulting in a column bed 1.5 cm in diameter and 46 cm in height.
      Note: The SEC column can be reused.
    16. The column is attached to the BioLogic LP System and equilibrated with 150-200 ml TBS at a flow rate of 2 ml/min.
    17. Protein solution is then loaded and eluted in a 65 ml of buffer at 0.5 ml/min at room temperature. Elution of protein was monitored by absorbance at 280 nm with the typical OD280 values of 0.01-0.2.
      SEC protocol:

      Period of time (min)
      Flow rate (ml/ min)
      Action
      0-40
      0.5
      Discard
      40-130
      0.5
      Collect 1.5 min per fraction (0.75 ml each fraction)
      130-200
      0.5
      Divert to waste

    18. The 1.5 ml fractions from the elution peak (approximately 75-90 min retention time) are collected and pooled as follows: fraction numbers 18-27 (pool I); 28-31 (pool II); 32-46 (pool III).
      Note: The pool II usually contains the Afp or its derivates with the least amount of cellular contaminants, while pool I and III contain less concentrated Afp particles with higher amounts of cellular contaminants. However, due to slight variations in column filling density, the fractions to be pooled have to be determined experimentally each time the column is repoured.
    19. The pooled SEC fractions containing Afp or its derivatives are centrifuged at 151,139 x g for one hour and 10 min at 4 °C in a Beckman Optima TM L-100K ultracentrifuge (fixed angle).
      Notes:
      1. It is helpful to label the outside of the tube where the pellet is likely to precipitate.
      2. The tube after the centrifugation has to be handled very carefully. Proceed to the next step immediately after the ultracentrifugation step to avoid disturbance of the pellet.
    20. After the centrifugation the supernatant is carefully removed using a pipette without disturbing the pellet but leaving approximatley 150-200 µl that was left enabbling the later resuspenssion of the pellet (Figure 1).


      Figure 1. Removing supernatant after ultracentrifugation. The supernatant is removed using a pipette; approximately 150-300 µl covering the invisible pellet is left behind.

    21. The tube is incubated for 30-60 min at 22 °C at 40 rpm, tilted with the labelled side down (Figure 2) in order to gently solubilise the pellet.


      Figure 2. Gentle resuspension of the pellet. The tube is placed as shown in the figure and gently agitated for 30-60 min.

    22. Following incubation, the often transparent retentate containing particles of interest is resuspended in residual buffer (usually approximately 150-200 μl) by pipetting up and down.
    23. The resuspended pellet is transferred to the 1.7 ml Eppendorf tube and centrifuged for 30 sec at 16,000 x g. The pellet is discarded and supernatant is transferred to a new eppendorf tube with 0.02% sodium azide (final concentration) added. This preparation is used for visualizing Afp particles or its variants using transmission electron microscopy or for other applications.
      Note: Afp particles of approximately 70% purity can be purified using this method.

  2. Negative staining and TEM of Afp and its variants
    1. The grids are charged by using glow discharging following manufacturer’s instructions no longer than 15 min before applying a sample.
    2. To visualise Afp and its variants, 3 μl of the re-suspended pellet is applied to a freshly glow-discharged plastic-coated 200-mesh grid.
    3. The sample is allowed to settle for 60 sec.
    4. Excess fluid is wicked off using Whatman #1 filter paper and then stained for 45 sec with a 0.7% filtered uranyl acetate (pH 5.0).
      Note: 0.5-1.0% can also be used.
    5. The grid is left to air dry on a filter paper for 5-15 min.
    6. Grids are examined in a Morgagni 268D TEM at a nominal magnification of 70,000-180,000 operated at 80 KeV following manufacturer’s instructions. The images are captured using a Megapixel III digital camera. Several examples of the Afp images are shown in Figure 3.


      Figure 3. Examples of the uncropped Afp images taken at nominal magnifications 89,000 (A) and 110,000 (B)

Recipes

  1. 20% L-Arabinose (5 ml stock solution)
    Mix 1 g Arabinose with 5 ml dH2O
  2. 0.4x LB (50 ml)
    Mix 20 ml LB and 30 ml dH2O
  3. 0. 2% sodium azide (10 ml stock solution)
    Mix 0.02 g sodium azide with 10 ml dH2O
  4. 250 mM TBS (pH 7.4) (500 ml 10x stock)
    Mix 40 g NaCl
    1 g KCl
    2.42 g Tris base
    16.5 g Tris-HCl
    With 490 ml dH2O
    Ajust pH to 7.5 with HCl (approximately 1 ml 6 M HCl)
    Add dH2O to 500 ml
    Use in 1:10 dilution (25 mM)
    Filter sterilize (0.45 μm) before using for SEC
  5. TM buffer
    Mix 2.52 g Tris-HCl with 3.25 g MgCl2
    Add dH2O to 800 ml and adjust pH to 7
    Autoclave and sterile filter (0.45 μm)

Acknowledgments

The protocol was adapted from Rybakova et al. (2013). We are grateful to the following colleagues from AgResearch: Dr Duane Harland for help with TEM, Sandra Jones for assisting in protein purification, Chikako van Koten for statistical assessments and Stefan Clerens for LC-MS/MS. Sam Yu (Izon Science, Christchurch, New Zealand) is thanked for the assessment of Afp particles. This research was funded by Grant C10X0804 of the New Economy Research Fund (NERF), administered by the New Zealand Foundation for Research, Science and Technology.

References

  1. Rybakova, D., Radjainia, M., Turner, A., Sen, A., Mitra, A. K. and Hurst, M. R. (2013). Role of antifeeding prophage (Afp) protein Afp16 in terminating the length of the Afp tailocin and stabilizing its sheath. Mol Microbiol 89(4): 702-714.

简介

噬菌体噬菌体抗原噬菌体(Afp)形成噬菌体尾样颗粒(太古霉素),其导致新西兰草禾本科(Costelytra zealandica)的饲养活性的停止。在这里,我们描述了Afp颗粒及其变体的更详细的纯化方案,其基于我们的原始出版物中描述的程序(Rybakova等人,2013)。纯化程序包括在阿拉伯糖诱导型启动子下诱导含有emp 基因的大肠杆菌(大肠杆菌)细胞24小时。收获细胞,在冰上超声处理,然后进行DNA酶处理和离心。然后将上清液过滤灭菌,并应用于尺寸排阻色谱(SEC)柱。合并含有Afp或其变体的级分并超速离心。除去上清液,将透明沉淀物重悬浮于残余缓冲液中。该程序产生约70%纯的Afp颗粒或其变体。使用透射电子显微镜(TEM)对Afp颗粒进行负染色并可视化。

材料和试剂

注意:除非另有说明,所有溶液均用蒸馏的去离子Millipore微过滤(MilliQ)水制备,在1.1kg/cm 2真空下高压灭菌15分钟,/121℃,并在室温下储存。如果要加入抗生素或补充剂,则在加入抗生素或补充溶液之前将培养基冷却至50-60℃的温度。以所述浓度(μg/ml)使用以下抗生素:氨苄青霉素,100;氯霉素,30;和壮观霉素,100

  1. E。在阿拉伯糖启动子下含有 afp 基因的大肠杆菌菌株 注意:请参考原始手稿(Rybakova等,2013)了解菌株和抗生素耐药性描述。
  2. LB-琼脂(Merck KGaA,目录号:VM459383236)
  3. DNase(Roche Diagnostics,目录号:04716728001)
  4. 0.7%过滤(孔径0.2um)在无菌水(pH5.0)(Electron Microscopy Sciences,目录号:22400)中稀释的乙酸双氧铀
  5. Sephacryl S-400 HR树脂(GE, http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/en/GELifeSciences/products/AlternativeProductStructure_17395/17060901
  6. 20%L-阿拉伯糖(Acros organics)(Thermo Fisher Scientific,目录号:AC104985000)(参见配方)
  7. 0.4x Luria-Bertani肉汤(LB)(Life Technologies,目录号:12795-027)(参见配方)
  8. 0.2%叠氮化钠(Sigma-Aldrich,目录号:S8032)(参见Recipes)
  9. 250 mM TBS缓冲液(见配方)
  10. TM缓冲区(参见配方)

设备

  1. 分光光度计
  2. 轨道混合器培养箱(Raytek Corporation)
  3. 1.7 ml微量离心管
  4. Eppendorf离心机
  5. Sanyo soniprep 150超声波仪(18Ω)(Panasonic Corporation)
  6. 0.45μm孔径的醋酸纤维素过滤器(Advantec MFS,目录号:03CP045AN)
  7. 50ml BD Falcon管
  8. 用于尺寸排阻色谱(SEC)的Sephacryl S-400 HR珠(GE Healthcare,目录号:17-0609-10)
  9. SEC柱,床体积为1.5×46cm(Bio-Rad Laboratories,型号:737-4155)
  10. 1.5×50cm(D×L)柱(Farmacia)
  11. BioLogic LP系统(Bio-Rad Laboratories,型号:731-8350)
  12. Beckman coulter Optima TM L-100K超速离心机(Beckman Coulter,型号:392050)
  13. Polaron E5100 SEM涂层装置
    注意:这个单位是从2000年之前,不再可用,但是类似的单位可以在 http://www.quorumtech.com/products/glow-discharge-system-free-standing.html
  14. Whatman#1滤纸(Whatman)
  15. 塑料涂覆的200目网格(ProSciTech,目录号:GCU200)
  16. Morgagni 268D TEM(FEI)
  17. 百万像素III数码相机
    注意:目录号和型号对于重现性不重要。
  18. 用于尺寸排阻色谱法的聚丙烯管(聚丙烯12×75mm,5ml)(Labserv,目录号:LBS504N)
  19. 用于超速离心的聚丙烯管(具有盖组件16×76mm,容量10.4ml的聚碳酸酯离心瓶)(Beckman Coulter,目录号:355603)
  20. Falcon管(Thermo Fisher Scientific,目录号:14-432-22)
  21. 保护微生物保存系统(技术服务顾问)在-80℃下保存大肠杆菌细胞
    注意:当需要时,从小瓶中取出单个接种的珠子,并滴入含有适当抗生素的LB肉汤中。

程序

注意:除非另有说明,离心步骤在4℃下进行。

  1. Afp纯化方案
    1. 使用来自具有大肠杆菌菌株的合适Afp或其衍生物的-80℃储备物的单个接种珠(保护微生物保存系统)来接种具有相应抗生素的3ml LB。 br />
    2. 将培养物在37℃下以250rpm搅拌温育过夜(过夜培养)
    3. 使用1ml过夜培养物接种具有适当抗生素的50ml新鲜LB。
    4. 将50ml细胞培养物在37℃下在搅拌下生长至OD 600的1.0。
      注意:OD 测量取自用LB培养基以1:10的比例稀释的细胞培养物。使用LB培养基的比色皿作为空白。
    5. 将细胞转移到无菌falcon管(50ml)中,并通过在室温下以2268×g离心10分钟收获。
    6. 将细胞沉淀重新悬浮在含有相应抗生素的新鲜50ml 0.4x LB肉汤中 注意:最初向每个沉淀中加入1ml的0.4x LB肉汤,并通过在管上剧烈的胶带将沉淀重新悬浮。然后用0.4x LB肉汤将管充满至50ml,并将管的内容物转移到与培养物生长相同的50ml烧瓶中。将抗生素直接加入烧瓶中。
    7. 将500μl无菌的过滤的20%阿拉伯糖(终浓度0.2%)加入到每个50ml培养物中以诱导蛋白质表达。
      注意:添加阿拉伯糖后,避免摇动培养物。
    8. 将培养物在22℃以40rpm再温育24小时。
    9. 通过在2268×g离心10分钟收获诱导的细胞。
      注意:如果沉淀物太松散,更高的离心速度(4,629×g,10分钟)可能是必需的。
    10. 将来自每50ml培养物的沉淀物在4℃下在0.8ml预冷的TM缓冲液中重悬浮
    11. 将得到的约0.9ml重悬浮液转移到1.7ml微量离心管中,并使用Sanyo Soniprep 150超声波仪(18Ω)进行三次20秒的超声处理。 在超声处理步骤之间包括在冰上至少1分钟的孵育
    12. 加入1μl(10单位)DNA酶,将样品在室温下放置10分钟。
    13. 在DNase温育步骤后,将悬浮液在4℃下以16,000×g离心10分钟。
    14. 弃去沉淀,通过0.45μm孔径的过滤器过滤含有可溶性重组蛋白的澄清上清液。
      注意:
      1. 如果样品需要储存1-2天,则将0.02%(终浓度)NaN 3加入每个管中。
      2. 不测试存储超过2天; 然而,为了更好的再现性,建议在该步骤后立即进行纯化。
    15. 根据制造商的说明书将树脂装入1.5×50cm(D×L)柱中,得到直径为1.5cm,高为46cm的柱床。 注意:SEC列可以重复使用。
    16. 将柱连接到BioLogic LP系统上,并以2ml/min的流速用150-200ml TBS平衡。
    17. 然后将蛋白质溶液加载并在室温下以0.5ml/min在65ml缓冲液中洗脱。 通过280nm的吸光度监测蛋白质的洗脱,典型的OD 280值为0.01-0.2。
      SEC协议:

      时间(分)
      流速(ml/min)
      行动
      0-40
      0.5
      舍弃
      40-130
      0.5
      每份收集1.5分钟(每份0.75ml)
      130-200
      0.5
      转移废物

    18. 收集来自洗脱峰(约75-90分钟保留时间)的1.5ml级分,并如下混合:级分18-27(泳道I); 28-31(池II); 32-46(池III)。
      注意:池II通常含有具有最少量细胞污染物的Afp或其衍生物,而池I和III含有较少浓度的具有较高量细胞污染物的Afp颗粒。然而,由于柱填充密度的轻微变化,每次回收柱时,要汇集的级分必须由实验确定。
    19. 将含有Afp或其衍生物的汇集的SEC级分在Beckman Optima TM L-100K超速离心机(固定角度)中以151,139×g离心1小时10分钟,4℃。
      注意:
      1. 有助于标记管的外部,其中沉淀可能沉淀。
      2. 离心后的管必须非常小心地处理。在超速离心步骤后立即进行下一步骤,以避免颗粒的干扰。
    20. 离心后,使用移液管小心地除去上清液,而不干扰沉淀,但留下大约150-200μl,留下使沉淀的后期重悬浮(图1)。


      图1.超速离心后除去上清液。使用移液管除去上清液;大约150-300μl覆盖了不可见的颗粒。

    21. 将管在22℃以40rpm孵育30-60分钟,用标记侧向下倾斜(图2),以轻轻地溶解沉淀。


      图2.温和的颗粒重悬。管如图所示放置,轻轻搅动30-60分钟。

    22. 孵育后,将含有感兴趣的颗粒的经常透明的保留物通过上下吹吸再悬浮于残余缓冲液(通常约150-200μl)中。
    23. 将重悬浮的沉淀转移到1.7ml Eppendorf管中,并以16,000×g离心30秒。弃去沉淀并将上清液转移至添加有0.02%叠氮化钠(终浓度)的新的埃彭道夫管中。该制剂用于使用透射电子显微镜或其它应用观察Afp颗粒或其变体。
      注意:约70%纯度的Afp颗粒可以使用此方法纯化。

  2. Afp及其变体的阴性染色和TEM
    1. 在施加样品之前,根据制造商的说明,使用辉光放电对栅极充电,不超过15分钟
    2. 为了显现Afp及其变体,将3μl重悬浮的沉淀物施加到新鲜辉光放电的涂覆有200目网格的塑料上。
    3. 使样品沉降60秒。
    4. 使用Whatman#1滤纸吸干过量流体,然后用0.7%过滤的乙酸铀酰(pH 5.0)染色45秒。
      注意:也可以使用0.5-1.0%。
    5. 将网格在滤纸上风干5-15分钟。
    6. 在标称放大率为70,000-180,000的Morgagni 268D TEM中,在80KeV下操作,根据制造商的说明书检查栅格。 使用百万像素III数码相机捕获图像。 Afp图像的几个示例如图3所示。


      图3.以标称放大倍数89,000(A)和110,000(B)拍摄的未裁剪Afp图像的示例

食谱

  1. 20%L-阿拉伯糖(5ml储备液) 将1g阿拉伯糖与5ml dH 2 O混合
  2. 0.4x LB(50ml) 将20ml LB和30ml dH 2 O混合
  3. 0.2%叠氮化钠(10ml储备液) 将0.02g叠氮化钠与10ml dH 2 O混合
  4. 250mM TBS(pH 7.4)(500ml 10×储备液) 混合40 g NaCl
    1克KCl
    2.42克Tris碱
    16.5g Tris-HCl
    使用490ml dH 2 O
    用HCl(约1ml 6M HCl)调节pH至7.5 将dH <2> O添加到500ml
    使用1:10稀释(25 mM)
    过滤灭菌(0.45μm)后用于SEC
  5. TM缓冲区
    将2.52g Tris-HCl与3.25g MgCl 2混合 将dH 2 O加入到800ml中并将pH调节至7 高压灭菌和无菌过滤器(0.45μm)

致谢

该方案改编自Rybakova等人(2013)。 我们感谢以下来自AgResearch的同事:Duane Harland博士帮助TEM,Sandra Jones帮助蛋白质纯化,Chikako van Koten进行统计学评估,Stefan Clerens为LC-MS/MS。 感谢Sam Yu(Izon Science,Christchurch,新西兰)对Afp颗粒的评估。 这项研究由新经济研究基金(NERF)的拨款C10X0804资助,由新西兰研究,科学和技术基金会管理。

参考文献

  1. Rybakova,D.,Radjainia,M.,Turner,A.,Sen,A.,Mitra,A.K.和Hurst,M.R。(2013)。 抗原噬菌体(Afp)蛋白Afp16在终止Afp太洋蛋白的长度并稳定其鞘的作用 。 Mol Microbiol 89(4):702-714。
  • 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: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Rybakova, D., Mitra, A. K. and Hurst, M. R. (2014). Purification and TEM of Afp and Its Variants. Bio-protocol 4(10): e1132. DOI: 10.21769/BioProtoc.1132.
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。