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Hydroxyproline-rich glycoproteins (HRGPs) are major protein components in dicot primary cell walls and generally account for more than 10% of the wall dry weight. As essential members of the HRGP superfamily, extensins (EXTs) presumably function in the cell wall by assembling into positively charged protein scaffolds (Cannon et al., 2008) that direct the proper deposition of other wall polysaccharides, especially pectins, to ensure correct cell wall assembly (Hall and Cannon, 2002; Lamport et al., 2011a). Extensins are recalcitrant to purification as they are rapidly cross-linked into a covalent network after entering the cell wall but there exists a short time window in which newly synthesized extensin monomers can be extracted (Smith et al., 1984; Smith et al., 1986) by salt elution. A detailed protocol for extraction of extensin and other wall structural proteins has been described earlier (Lamport et al., 2011b). The protocol elaborated here provides an approach to studying the self-assembly of extensins and potentially of other cell wall components in vitro using AFM.

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Atomic Force Microscopy (AFM) Analysis of Cell Wall Structural Glycoproteins in vitro
原子力显微镜(AFM)法体外分析细胞壁的结构糖蛋白

植物科学 > 植物细胞生物学 > 细胞成像
作者: Yuning Chen
Yuning ChenAffiliation: Department of Chemistry and Biochemistry, Ohio University, Athens, USA
For correspondence: yuc27@psu.edu
Bio-protocol author page: a2380
Liwei Chen
Liwei ChenAffiliation: Suzhou Institute of Nano-Tech and Nano-Bionics (SINANO), Chinese Academy of Science, Suzhou, China
Bio-protocol author page: a2381
Marcia J. Kieliszewsk
Marcia J. KieliszewskAffiliation: Department of Chemistry and Biochemistry, Ohio University, Athens, USA
Bio-protocol author page: a2382
 and Maura C. Cannon
Maura C. CannonAffiliation: Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst, USA
Bio-protocol author page: a2383
Vol 5, Iss 14, 7/20/2015, 1918 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1534

[Abstract] Hydroxyproline-rich glycoproteins (HRGPs) are major protein components in dicot primary cell walls and generally account for more than 10% of the wall dry weight. As essential members of the HRGP superfamily, extensins (EXTs) presumably function in the cell wall by assembling into positively charged protein scaffolds (Cannon et al., 2008) that direct the proper deposition of other wall polysaccharides, especially pectins, to ensure correct cell wall assembly (Hall and Cannon, 2002; Lamport et al., 2011a). Extensins are recalcitrant to purification as they are rapidly cross-linked into a covalent network after entering the cell wall but there exists a short time window in which newly synthesized extensin monomers can be extracted (Smith et al., 1984; Smith et al., 1986) by salt elution. A detailed protocol for extraction of extensin and other wall structural proteins has been described earlier (Lamport et al., 2011b). The protocol elaborated here provides an approach to studying the self-assembly of extensins and potentially of other cell wall components in vitro using AFM.
Keywords: Plant cell wall(植物细胞壁), Structural glycoprotein(结构糖蛋白), Extensin(伸展蛋白), Self-assembly(自组装), Atomic force microscope(原子力显微镜)

[Abstract]

Materials and Reagents

  1. Monomeric extensin proteins extracted from different plant cell suspension culture lines. For a detailed extensin extraction protocol see Lamport et al. (2011b).
  2. Bovine serum albumin (BSA, lyophilized powder, crystallized, purity ≥ 98.0%) (Sigma-Aldrich, catalog number: 05470 )
  3. Sodium acetate (NaOAc, anhydrous, purity ≥ 99.0%) (Sigma-Aldrich, catalog number: S8750 )
  4. Acetic acid (HOAc, ACS reagent, purity ≥ 99.7%) (Sigma-Aldrich, catalog number: 320099 )
  5. Deionized-distilled water (ddH2O)
  6. 75% ethanol (prepared from 100% ethanol, ACS reagent, purity ≥ 99.5%) (Sigma-Aldrich, catalog number: 459844 )
  7. Loctite® Epoxy instant mix glue
  8. 50 mM NaOAc buffer (see Recipes)
  9. 75% ethanol (see Recipes)

Equipment

  1. Highly ordered pyrolytic graphite (square shape 5 mm (L) x 5 mm (W) x 1 mm (H) in dimension) (Structure Probe, catalog number: 476HPAB )
  2. Kimwipes (VWR International, catalog number: 470173498 )
  3. Plastic petri dishes (100 x 15 mm dimension) (VWR International, catalog number: 25384302 )
  4. NCS18 silicon probe (75 kHz, 3.5 N/m) (Mikromasch, catalog number: HQ:NSC18/Cr-Au-15)
  5. Scotch tape (single sided Scotch® MagicTMTape 810)
    Note: Available from office supply stores like Staples.
  6. 10 ml sterile syringes (BD Biosciences, catalog number: 309659 )
  7. 0.2 µm syringe filters (Whatman, catalog number: 6896-2502 )
  8. 20 ml disposable scintillation glass vials (VWR International, catalog number: 66022065 )
  9. Eppendorf tubes(1.5 ml) (Eppendorf, catalog number: 022431081 )
  10. Regular microscope slides (dimension 25 x 75 x 1mm)
  11. MFP-3D-SA AFM (Asylum Research, model: MFP-3D-SA AFM)
  12. Stainless steel tweezers (Sigma-Aldrich, catalog number: T5415 )
  13. Cylinder of nitrogen gas with a pressure regulator and nozzle
  14. Vortex mixer
  15. Centrifuge (to fit Eppendorf 1.5 ml tubes)

Software

  1. IGOR Pro 6 (WaveMetrics, Portland, OR)

Procedure

  1. Preparations before imaging
    1. Buffer preparation
      1. Filter ddH2O through 0.2 µm syringe filter into clean glass vials before use.
      2. Prepare 50 mM NaOAc buffer (pH 5.2). Filter buffer through 0.2 µm syringe filter into clean glass vials before use.
    2. Prepare protein stock solutions
      Dissolve protein samples to a concentration of 1 mg/ml in filtered buffer as stock solutions using 1.5 ml Eppendorf Protein LoBind Tube (see Note 1). Aliquot stock solutions into 100 µl fractions (see Note 2). Store stock aliquots at -20 °C before use.
    3. Preparation of imaging “substrate” (see Note 3)
      Clean one glass slide with ddH2O followed by rinsing with 75% ethanol. Air dry the slide and apply the Loctite® Epoxy instant mix glue to the center of the slide. After a quick mix of the two glue components, carefully remove the HOPG from its case using tweezers and place it on top of the glue. Allow overnight incubation for the tight binding of HOPG to the slide.

  2. Sample preparation for AFM imaging
    1. Prepare diluted protein samples
      Dilute protein stocks to desired concentration (see Note 4) with corresponding buffer or ddH2O using Eppendorf Protein LoBind Tubes (see Note 1), mix the dilutes by gentle vortexing and then spin down the protein solutions at 3,000 x g for 2 min.
    2. Substrate preparation
      HOPG has multiple layers and each layer is atomically flat. Carry out each AFM experiment on a fresh layer. To obtain a new layer of HOPG, simply use Scotch tape to adhere to the previous surface and peel off the old surface. Note that sometimes multiple attempts are required to achieve a visually flat HOPG surface (see Note 5).
    3. Protein binding to HOPG
      After obtaining the fresh HOPG surface, place the glass slide with HOPG in a petri dish and deposit 100 µl of protein solution onto the center of the HOPG surface. Cover the petri dish with lid to prevent unnecessary contamination from the surrounding environment. Place moist Kimwipes in the petri dish (avoid direct contact with HOPG) if long incubation time (more than 30 min) is needed to reduce sample evaporation.
      Allow the protein solution to incubate on HOPG for one minute or longer (see Note 6) at room temperature. Following incubation, use Kimwipes (see Note 7) to blot away the protein solution and rinse the HOPG surface with 100 µl ddH2O. Blot away the remaining liquid and dry the surface further under a slow steam of N2 gas flow for 2 min. At this point, the sample preparation is complete and the HOPG surface is ready for AFM scanning.

  3. AFM imaging of the self-assembly of extensins
    Refer to the user’s manual (http://mmrc.caltech.edu/Asylum/Asylum%20MRP-3D%20manual.pdf) from Asylum Research for the operation of the MFP-3D SA AFM. Use “AC mode” (Chapter 6 in the manual, pp 121-142) for the scanning of samples in air. The probe we used was a NCS18 silicon probe from Mikromasch (see Materials and Reagents). For better resolution, Hi'Res-C14 probes (catalog number: Hi'Res-C14/Cr-Au-5) are recommended because of their finer tip radius (~1 nm).
    After probe installation, optical detection laser alignment and probe tuning, on the master panel (see pp136 in the manual), select the initial scan parameters as follows:
    Scan Size = 5.00 µm
    Scan Rate = 0.75 Hz (see Note 8)
    Scan Points/Lines = 256
    Set Point = 800.00 mV
    Integral Gain = 4 (see Note 9)
    Engage the AFM probe to sample surface by lowering the AFM head and start scanning. Here note that the “Drive Amplitude/Frequency” values on the master panel are the values automatically filled in by the instrument upon the finishing of probe tuning, thus no changes of these values are needed.
    After the start of scanning, first allow the system to scan for 2 min to stabilize the probe and adjust the instrument to surrounding environmental vibrations. Then manually lower the “Set Point” value until surface features start to appear on the height image. Imaging optimization can be achieved by adjusting “Set Point” and “Integral Gain” values. Finally, the “Scan Points” and “Scan Lines” can be increased to 512 or even 1,024 (see Note 10) for more pixels in each image thus enhance the image quality.
    For the determination of self-assembly pattern, take at least five images at five different locations on the HOPG surface for each sample.

  4. Image processing
    Images need to be flattened using the “flatten” function in IGOR Pro software before any measurements. Information of height (i.e. the diameter) and length of a molecule can be directly measured on the images using the IGOR Pro software. The default color setting of the images is grey but IGOR Pro has a variety of built-in color schemes that can be used to false-color images afterwards. In the meantime, the contrast and brightness of each image can also be adjusted for a better presentation of the image.

Representative data



Figure 1. Images of extensin self-assembly. (a) Monomeric extensin precursor 1 from tomato suspension culture (TOMP1). (b) Extensin analog YK8. (c) Extensin analog FK9. (d) BSA used as a control. YK8 and FK9 are extensin analogs that contain 8 repeats of SOOOOSOSOOOOYYYK and 9 repeats of SOOOOSOSOOOOFFFK in their protein sequences, respectively, and are purified from tobacco BY2 cells in culture (Held et al., 2004). One letter amino acids codes: Tyrosine (Y), Lysine (K), Phenylalanine (F), Serine (S), Hydroxyproline (O). All proteins were imaged at 10 μg/ml in pH 5.2 NaOAc buffer, following deposition for 1 min. The white scale bar: 500 nm. TOMP1 and the extensin analogs all showed a head to tail dendritic self-assembly similar to that of EXTENSIN 3 from Arabidopsis (Cannon et al., 2008), while BSA showed no such assembly but only protein aggregation. Red arrow in image (d) indicates the scattered single sphere shaped BSA molecules.


Figure 2. Concentration and deposition time dependent self-assembly of TOMP1 in pH 5.2 NaOAc buffer. a. TOMP1 showed head to tail dendritic self-assembly after 10 min deposition on HOPG at 5 µg/ml. b. At a higher concentration (10 µg/ml) TOMP1 assembled into a porous network in the same time period, indicating the extent of self-assembly is concentration dependent. c. Similar assembly of TOMP1 as in (a), highlighted by the white circles, was observed when 10 µg/ml of TOMP1 was deposited for 1 min, indicating that the extent of TOMP1 self-assembly also followed a time dependent manner. Examples of measurements for TOMP1 self-assembly: (1) The average segment length, highlighted by white bars in the circles in image (c), is 78.2 ± 4.2 nm this is in agreement with previously electron microscopy measurements of the TOMP1 polypeptide length at about 79 nm (Heckman et al., 1988). This observation indicates one TOMP1 molecule occurs in each segment. (2) The average single molecule height, highlighted by red bars in image (c), is 2.7 ± 0.2 nm. This value corresponds to the diameter of single TOMP1 molecule. (3) The average height of the connecting points for three molecules, highlighted by green circles in image (c), is 5.2 ± 0.2 nm. This value indicates that the TOMP1 molecules form lateral and overlapping associations up to two molecules deep. The white scale bar: 500 nm

Notes

  1. The protein concentrations used in stock and imaging are relatively low. The use of Eppendorf Protein LoBind tubes will prevent non-specific binding of protein to the tubes thus resulting in a more accurate protein amount used in experiments.
  2. Repeated freezing-thawing will lead to protein degradation and unexpected aggregation. Use one aliquot per experiment to help prevent such damage to the protein stocks.
  3. In AFM imaging, the surface on which sample solutions are deposited is called “substrate”. We used HOPG for imaging of proteins due to its hydrophobicity, which allows the stable binding of proteins to the surface. To study other wall components, for instance wall polysaccharides and cell wall itself, other substrates such as mica (for polysaccharides) or charged glass slides (for cell wall) can be used as alternatives to ensure the binding of sample to the substrate surface.
  4. Depending on the purpose of the experiments, various protein concentrations can be used. From our experience, a low protein concentration (5 or 10 µg/ml) will benefit in the observation of extensin single molecular assembly while a higher concentration (50 µg/ml) will result in an orderly formed network.
  5. HOPG will get thinner after multiple uses, thus making it progressively more difficult to obtain a new visually flat surface. Based on its original height dimension (1 mm), a replacement is recommended when the HOPG reaches less than 0.4 mm in height.
  6. Similar to Note 4, the length of incubation time also determines the pattern of observed self-assembly. Usually after longer incubation, a well-formed extensin network is observed. From our experience, the attachment of extensin molecules to the HOPG surface happens quickly. Incubation time as short as 1 min is sufficient for the binding of extensins to HOPG.
  7. The use of regular paper towels is not recommended since there might be unexpected fibers or debris that might contaminate the sample surface.
  8. Use a slow scan rate at first to protect the probe, the “Scan Speed” is automatically set upon the selection of “Scan Rate”.
  9. Use a low “Integral Gain” in the beginning to protect the probe. This value can be elevated in a later scan for better image quality.
  10. “Scan Points” and “Scan Lines” determine the number of pixel point on each line and the number of lines scanned in the image, in most of the cases these two values should be kept the same.

Recipes

  1. 50 mM NaOAc buffer
    0.41 g NaOAc
    100 ml distilled water
    Adjust pH to 5.2 with HOAc
  2. 75% ethanol
    Add 25 ml water to 75 ml 100% ethanol to make 100 ml 75% ethanol

Acknowledgements

This protocol was adapted with modification from Cannon et al. (2008). Funding of this work was from National Science Foundation (#IOS955569 to M.J.K and IOS0955805 to M.C.C).

References

  1. Cannon, M. C., Terneus, K., Hall, Q., Tan, L., Wang, Y., Wegenhart, B. L., Chen, L., Lamport, D. T., Chen, Y. and Kieliszewski, M. J. (2008). Self-assembly of the plant cell wall requires an extensin scaffold. Proc Natl Acad Sci USA 105(6): 2226-2231.
  2. Hall, Q. and Cannon, M. C. (2002). The cell wall hydroxyproline-rich glycoprotein RSH is essential for normal embryo development in Arabidopsis. Plant Cell 14(5): 1161-1172.
  3. Heckman, J. W., Terhune, B. T. and Lamport, D. T. (1988). Characterization of native and modified extensin monomers and oligomers by electron microscopy and gel filtration. Plant Physiol 86(3): 848-856.
  4. Held, M. A., Tan, L., Kamyab, A., Hare, M., Shpak, E. and Kieliszewski, M. J. (2004). Di-isodityrosine is the intermolecular cross-link of isodityrosine-rich extensin analogs cross-linked in vitro. J Biol Chem 279(53): 55474-55482.
  5. Lamport, D. T., Kieliszewski, M. J., Chen, Y. and Cannon, M. C. (2011a). Role of the extensin superfamily in primary cell wall architecture. Plant Physiol 156(1): 11-19.
  6. Lamport, D. T., Tan, L. and Kieliszewski, M. J. (2011b). Structural proteins of the primary cell wall: extraction, purification, and analysis. Methods Mol Biol 715: 209-219.
  7. Smith, J. J., Muldoon, E. P. and Lamport, D. T. A. (1984). Isolation of extensin precursors by direct elution of intact tomato cell suspension cultures. Phytochem 23, 1233-1239.
  8. Smith, J. J., Muldoon, E. P., Willard, J. J. et al. (1986). Tomato extensin precursors P1 and P2 are highly periodic structures. Phytochem 5, 1021-1030.

材料和试剂

  1. 单体延伸蛋白从不同植物细胞悬浮培养基中提取。 关于详细的延伸提取方案,参见Lamport等人(2011b)。
  2. 牛血清白蛋白(BSA,冻干粉,结晶,纯度≥98.0%)(Sigma-Aldrich,目录号:05470)
  3. 乙酸钠(NaOAc,无水,纯度≥99.0%)(Sigma-Aldrich,目录号:S8750)
  4. 乙酸(HOAc,ACS试剂,纯度≥99.7%)(Sigma-Aldrich,目录号:320099)
  5. 去离子蒸馏水(ddH 2 O)
  6. 材料和试剂

    1. 单体延伸蛋白从不同植物细胞悬浮培养基中提取。 关于详细的延伸提取方案,参见Lamport等人(2011b)。
    2. 牛血清白蛋白(BSA,冻干粉,结晶,纯度≥98.0%)(Sigma-Aldrich,目录号:05470)
    3. 乙酸钠(NaOAc,无水,纯度≥99.0%)(Sigma-Aldrich,目录号:S8750)
    4. 乙酸(HOAc,ACS试剂,纯度≥99.7%)(Sigma-Aldrich,目录号:320099)
    5. 去离子蒸馏水(ddH 2 O)
    6. ...
    7. Kimwipes (VWR International, catalog number: 470173498)
    8. Plastic petri dishes (100 x 15 mm dimension) (VWR International, catalog number: 25384302)
    9. NCS18 silicon probe (75 kHz, 3.5 N/m) (Mikromasch, catalog number: HQ:NSC18/Cr-Au-15)
    10. Scotch tape (single sided Scotch® MagicTMTape 810)
      Note: Available from office supply stores like Staples.
    11. 10 ml sterile syringes (BD Biosciences, catalog number: 309659)
    12. 0.2 µm syringe filters (Whatman, catalog number: 6896-2502)
    13. 20 ml disposable scintillation glass vials (VWR International, catalog number: 66022065)
    14. Eppendorf tubes(1.5 ml) (Eppendorf, catalog number: 022431081)
    15. Regular microscope slides (dimension 25 x 75 x 1mm)
    16. MFP-3D-SA AFM (Asylum Research, model: MFP-3D-SA AFM)
    17. Stainless steel tweezers (Sigma-Aldrich, catalog number: T5415)
    18. Cylinder of nitrogen gas with a pressure regulator and nozzle
    19. 涡流搅拌器
    20. 离心机(以配合Eppendorf 1.5ml管)

    软件

    1. IGOR Pro 6(WaveMetrics,Portland,OR)

    程序

    1. 成像前准备
      1. 缓冲液准备
        1. 在使用前将ddH 2 O通过0.2μm注射器过滤器过滤到干净的玻璃小瓶中
        2. 制备50mM NaOAc缓冲液(pH 5.2)。 在使用前,通过0.2μm注射器过滤器将缓冲液过滤到干净的玻璃小瓶中。
      2. 准备蛋白储备液
        溶解蛋白质样品至1mg/ml的浓度,过滤 缓冲液作为储备溶液,使用1.5ml Eppendorf Protein LoBind Tube (见注1)。 将储备溶液分成100μl部分(见注释 2)。 使用前将储备液等分试样储存在-20°C
      3. 成像"基板"的准备(见注3)
        用ddH 2 O洗涤一个载玻片,然后用75%乙醇冲洗。 空气干燥载玻片,并将Loctite ®环氧速溶混合胶水涂抹 幻灯片的中心。 在快速混合两种胶组分后, 使用镊子小心地从其盒中移除HOPG并将其放置 顶部的胶水。 允许过夜孵育的紧密结合 HOPG到幻灯片。

    2. AFM成像的样品准备
      1. 准备稀释的蛋白质样品
        稀释蛋白储备到所需 浓度(参见注释4)与相应的缓冲液或ddH 2 O使用 Eppendorf Protein LoBind Tubes(见注1),轻轻混合稀释液 涡旋,然后将蛋白质溶液以3000×g离心2分钟 min。
      2. 基材准备
        HOPG有多层,每层 层是原子平坦的。 每天进行一次AFM实验 层。 要获得一个新的HOPG层,只需使用苏格兰磁带粘附 到先前的表面并剥离旧表面。 注意 有时需要多次尝试以实现视觉上平坦的HOPG   表面(见注5)。
      3. 蛋白结合HOPG
        后 获得新鲜的HOPG表面,将带有HOPG的载玻片放在a中 培养皿和沉积100微升的蛋白质溶液到中心   HOPG表面。 用盖子盖住培养皿,以防止不必要 污染从周围环境。 放置潮湿Kimwipes 如果培养时间长,培养皿(避免与HOPG直接接触) (大于30分钟)以减少样品蒸发。
        允许 将蛋白质溶液在HOPG上孵育1分钟或更长时间 注6)。 孵育后,使用Kimwipes(见 注7)吸去蛋白质溶液并冲洗HOPG表面 与100μlddH 2 O。 刮除剩余的液体并干燥表面 进一步在N 2气流的缓慢蒸汽下2分钟。 在这一点上, 样品制备完成,HOPG表面准备用于AFM 扫描。

    3. 延伸素自组装的AFM成像
      请参阅用户手册( http://mmrc.caltech.edu/Asylum) /Asylum%20MRP-3D%20manual.pdf ),来自Asylum Research的MFP-3D SA AFM的操作。使用"AC模式"(手册第6章,第121-142页)在空气中扫描样品。我们使用的探针是来自Mikromasch的NCS18硅探针(参见材料和试剂)。为了更好的分辨率,推荐使用Hi'Res-C14探头(目录号:Hi'Res-C14/Cr-Au-5),因为它们的尖端半径更小(〜1 nm)。 探头安装后,光学检测激光对准和探头调谐,在主面板上(参见手册中的第136页),选择初始扫描参数如下:
      扫描尺寸= 5.00μm
      扫描速率= 0.75 Hz(见注8)
      扫描点数/行数= 256
      设置点= 800.00 mV
      积分增益= 4(见注9)
      通过降低AFM头并开始扫描,将AFM探头接合到样品表面。请注意,主面板上的"驱动幅度/频率"值是在探头调整完成后仪器自动填充的值,因此不需要更改这些值。
      扫描开始后,首先让系统扫描2分钟,以稳定探针,并调整仪器以适应周围环境的振动。然后手动降低"设置点"值,直到表面要素开始显示在高度图像上。通过调整"设置点"和"积分增益"值可以实现成像优化。最后,对于每个图像中的更多像素,"扫描点"和"扫描线"可以增加到512或甚至1,024(见注10),从而提高图像质量。
      对于自组装图案的确定,对于每个样品在HOPG表面上的五个不同位置取至少五个图像。

    4. 图像处理
      在进行任何测量之前,需要使用IGOR Pro软件中的"平展"功能对图像进行平整。可以使用IGOR Pro软件在图像上直接测量分子的高度(即直径)和长度的信息。图像的默认颜色设置为灰色,但IGOR Pro具有各种内置颜色方案,可用于之后对图像进行假彩色。同时,也可以调整每个图像的对比度和亮度以更好地呈现图像

    代表数据



    图1.拉伸自组装的图像。(a)来自番茄悬浮培养(TOMP1)的单体延伸蛋白前体1。 (b)Extensin类似物YK8。 (c)Extensin类似物FK9。 (d)BSA用作对照。 YK8和FK9是分别在其蛋白质序列中含有8个重复的SOOOOSOSOOOOYYYK和9个重复的SOOOOSOSOOOOFFFK的伸展蛋白类似物,并且从培养物中的烟草BY2细胞中纯化(Held等人,2004)。单字母氨基酸代码:酪氨酸(Y),赖氨酸(K),苯丙氨酸(F),丝氨酸(S),羟脯氨酸(O)。所有蛋白质在pH 5.2 NaOAc缓冲液中以10μg/ml成像,沉积1分钟。白色比例尺:500nm。 TOMP1和伸展蛋白类似物都显示与来自拟南芥的EXTENSIN 3相似的头对尾树突自组装(Cannon等人,2008),而BSA显示没有这样的组装但仅显示蛋白质聚集。图像(d)中的红色箭头表示分散的单球形BSA分子

    图2.在pH 5.2 NaOAc缓冲液中TOMP1的浓缩和沉积时间依赖性自组装。 TOMP1在5μg/ml的HOPG上10分钟沉积后显示头对尾树突自组装。 b。在更高 浓度(10μg/ml)TOMP1在相同的时间段组装成多孔网络,表明自组装的程度是浓度依赖性的。 C。当10μg/ml的TOMP1沉积1分钟时,观察到类似的(a)中TOMP1的装配,用白色圆圈突出显示,表明TOMP1自组装的程度也遵循时间依赖性方式。 TOMP1自组装的测量的实例:(1)由图像(c)中的圆圈中的白色条突出的平均链段长度为78.2±4.2nm,这与之前的TOMP1多肽长度的电子显微镜测量一致约79nm(Heckman等人,1988)。该观察结果表明在每个区段中出现一个TOMP1分子。 (2)由图像(c)中的红色条突出显示的平均单分子高度为2.7±0.2nm。该值对应于单个TOMP1分子的直径。 (3)图像(c)中由绿色圆圈突出显示的三个分子的连接点的平均高度为5.2±0.2nm。该值表明TOMP1分子形成横向和重叠结合达两个分子深。白色比例尺:500nm

    笔记

    1. 库存和成像中使用的蛋白质浓度相对较低。使用Eppendorf蛋白LoBind管将防止蛋白质与管的非特异性结合,从而导致实验中使用更准确的蛋白质量。
    2. 重复冻融将导致蛋白质降解和意想不到的聚集。每个实验使用一个等分试样,以帮助防止这种蛋白原料的损害
    3. 在AFM成像中,其上沉积样品溶液的表面称为"基板"。我们使用HOPG蛋白质的成像,由于其疏水性,允许蛋白质与表面的稳定结合。为了研究其它壁组分,例如壁多糖和细胞壁本身,可以使用其它底物如云母(用于多糖)或带电载玻片(用于细胞壁)作为替代物,以确保样品与底物表面的结合。 br />
    4. 根据实验的目的,可以使用各种蛋白质浓度。从我们的经验,低蛋白浓度(5或10微克/毫升)将有利于观察拉伸单分子组装,而较高的浓度(50微克/毫升)将导致有序形成的网络。
    5. HOPG在多次使用后将变薄,因此使得逐渐更难以获得新的视觉上平坦的表面。根据其原始高度尺寸(1 mm),当HOPG高度小于0.4 mm时,建议更换。
    6. 类似于注释4,孵育时间的长度也决定了观察到的自组装的模式。通常在更长的温育后,观察到形成良好的延伸蛋白网络。从我们的经验,延伸素分子附着到HOPG表面发生很快。孵育时间短至1分钟就足以结合延伸素与HOPG
    7. 不推荐使用普通纸巾,因为可能有意想不到的纤维或碎片可能污染样品表面。
    8. 首先使用慢速扫描速率保护探头,"扫描速度"会在选择"扫描速率"时自动设置。
    9. 在开始使用低"积分增益"保护探头。 此值可以在以后的扫描中提高以获得更好的图像质量。
    10. "扫描点"和"扫描线"确定每行上的像素点数和在图像中扫描的行数,在大多数情况下,这两个值应保持不变。

    食谱

    1. 50mM NaOAc缓冲液 0.41g NaOAc
      100ml蒸馏水
      用HOAc调节pH至5.2
    2. 75%乙醇 向75ml 100%乙醇中加入25ml水,制成100ml 75%乙醇

    致谢

    该方案采用来自Cannon等人(2008)的修改。 这项工作的资金来自国家科学基金会(#IOS955569到M.J.K和IOS0955805到M.C.C)。

    参考文献

    1. Cannon,M.C.,Terneus,K.,Hall,Q.,Tan,L.,Wang,Y.,Wegenhart,B.L.,Chen,L.,Lamport,D.T.,Chen,Y.and Kieliszewski, 植物细胞壁的自组装需要一个延伸蛋白支架。 Proc Natl Acad Sci USA 105(6):2226-2231。
    2. Hall,Q.and Cannon,M.C。(2002)。 细胞壁富含羟脯氨酸的糖蛋白RSH对于拟南芥中的正常胚胎发育是必需的。植物细胞 14(5):1161-1172。
    3. Heckman,J.W.,Terhune,B.T.and Lamport,D.T。(1988)。 通过电子显微镜和凝胶过滤表征天然和修饰的延伸蛋白单体和寡聚物。 em> Plant Physiol 86(3):848-856。
    4. Held,M.A.,Tan,L.,Kamyab,A.,Hare,M.,Shpak,E。和Kieliszewski,M.J。(2004)。 二 - 异酪氨酸是交联的富含异酪氨酸的延伸蛋白类似物的分子间交联in vitro 。 J Biol Chem 279(53):55474-55482。
    5. Lamport,D.T.,Kieliszewski,M.J.,Chen,Y.and Cannon,M.C。(2011a)。 延伸超家族在原代细胞壁结构中的作用植物生理学 156(1):11-19。
    6. Lamport,D.T.,Tan,L。和Kieliszewski,M.J。(2011b)。 原代细胞壁的结构蛋白:提取,纯化和分析。 Methods Mol Biol 715:209-219
    7. Smith,J.J.,Muldoon,E.P。和Lamport,D.T.A。(1984)。 通过直接洗脱完整的番茄细胞悬浮培养物来分离外显子前体。 Phytochem 23,1233-1239
    8. Smith,J.J.,Muldoon,E.P.,Willard,J.J。等人(1986)。 番茄延伸蛋白前体P1和P2是高度周期性结构。 Phytochem 5 ,1021-1030。
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How to cite this protocol: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Chen, Y., Chen, L., Kieliszewsk, M. J. and Cannon, M. C. (2015). Atomic Force Microscopy (AFM) Analysis of Cell Wall Structural Glycoproteins in vitro. Bio-protocol 5(14): e1534. DOI: 10.21769/BioProtoc.1534; Full Text
  2. Cannon, M. C., Terneus, K., Hall, Q., Tan,L., Wang, Y., Wegenhart, B. L., Chen, L., Lamport, D. T., Chen, Y. andKieliszewski, M. J. (2008). Self-assemblyof the plant cell wall requires an extensin scaffold. Proc Natl Acad Sci USA 105(6): 2226-2231.




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