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Oct 2017

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α-Synuclein Aggregation Monitored by Thioflavin T Fluorescence Assay
通过硫磺素T荧光测定法监测核突触蛋白聚集   

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Abstract

Studying the aggregation of amyloid proteins like α-synuclein in vitro is a convenient and popular tool to gain kinetic insights into aggregation as well as to study factors (e.g., aggregation inhibitors) that influence it. These aggregation assays typically make use of the fluorescence dye Thioflavin T as a sensitive fluorescence reporter of amyloid fibril formation and are conducted in a plate-reader-based format, permitting the simultaneous screening of multiple samples and conditions. However, aggregation assays are generally prone to poor reproducibility due to the stochastic nature of fibril nucleation and the multiplicity of modulating factors. Here we present a simple and reproducible protocol to study the aggregation of α-synuclein in a plate-reader based assay.

Keywords: Amyloid (淀粉样蛋白), Aggregation (聚合), α-Synuclein (α-Synuclein), Thioflavin T assay (硫磺素T检测), Parkinson’s disease (帕金森病)

Background

Aggregation of endogenous proteins to amyloid fibrils is a pathogenic process that is associated with several disorders, e.g., neurodegenerative diseases like Alzheimer’s disease (AD) or Parkinson’s disease (PD) as well as systemic diseases like AL amyloidosis (Knowles et al., 2014). This process can be recapitulated in vitro in a plate-reader-based setup by aggregation assays based on Thioflavin T fluorescence, allowing the aggregation kinetics of amyloid proteins to be studied in dependence of various influencing factors.

Thioflavin T (ThT) is a fluorescence dye that has first been used for staining amyloid fibrils in histological samples by Vassar and Culling in 1959 (Vassar and Culling, 1959), its application for detecting and quantifying amyloid fibrils in vitro has first been described by Naiki et al. in 1989 (Naiki et al., 1989). Upon binding within the cross-β-architecture of amyloid fibrils, ThT changes its spectral characteristics (bathochromic wavelength shift to λex: 450 nm and λem: 482 nm) and exhibits a strong increase in fluorescence emission (Biancalana and Koide, 2010). It is therefore a very sensitive indicator of amyloid fibril formation and has been adapted to aggregation assays with synthetically and recombinantly produced amyloidogenic proteins, like the AD-associated protein amyloid-β (LeVine, 1993) as well as the PD-associated protein α-synuclein (Hashimoto et al., 1998).

Aggregation assays with Thioflavin T are nowadays mainly performed in fluorescence plate readers, where e.g., 96 conditions can be tested simultaneously. These assays suffer from poor reproducibility resulting from the stochastic nature of fibril nucleation and the multiplicity of factors affecting protein aggregation. Therefore, strategies to increase the reproducibility of ThT assays have been employed, such as the use of orbital shaking of the well plate during the measurement as well as the addition of glass beads to the wells to improve mixing (Giehm and Otzen, 2010).

Here, we describe a simple protocol for α-synuclein aggregation assays using ThT that comprises the following strategies to improve reproducibility and convenience:

  1. The use of N-terminally acetylated α-synuclein, which is the native state of the protein (Anderson et al., 2006). N-terminal acetylation of α-synuclein also increases reproducibility of aggregation half times in ThT assays (Iyer et al., 2016).
  2. Thirty seconds of shaking prior to the ThT fluorescence measurement leads to more reproducible fluorescence readings due to a more homogenous distribution of fibrils within the sample, including aggregation nuclei that preferentially form at the air-water-interface (Campioni et al., 2014).
  3. Glass beads (Ø 2.85-3.45 mm) are added to each well to improve mixing and homogeneity of the sample as described (Giehm and Otzen, 2010).
  4. We use half-area 96-well-plates (Corning, USA) that have a non-binding surface. Half-area wells save sample volume, as only 100-120 μl is needed per well. The non-binding surface e.g., prevents adsorption of amyloid fibrils to the well surface, a process that can cause aberrant readings in ThT assays (Murray et al., 2013).

Materials and Reagents

  1. 50 ml Falcon centrifuge tubes (TPP Techno Plastic Products, catalog number: 91050 )
  2. Sterile syringe filter, Filtropur S, 0.2 μm (SARSTEDT, catalog number: 83.1826.001 )
  3. Slide-A-LyzerTM dialysis cassettes 10 kDA MW cutoff, 3-12 ml (Thermo Fisher Scientific, catalog number: 66810 )
  4. HiTrap Q HP IEC column, 5 ml (GE Healthcare, catalog number: 17115401 )
  5. Superdex 75 16/60 SEC column, ~120 ml volume (GE Healthcare, catalog number: 28989333 )
  6. Protein LoBind Tubes 1.5 ml (Eppendorf, catalog number: 0030108116 )
  7. 96-well plates (Corning half-area, black and clear flat bottom, non-binding surface) (Corning, catalog number: 3881 )
  8. Sealing tape, clear polyolefin (Thermo Fisher Scientific, catalog number: 232701 )
  9. E. coli BL21DE3 competent cells (e.g., available from Merck, catalog number: 69450 )
  10. α-Synuclein in pT7-7 vector (e.g., Addgene, catalog number: 36046 )
    We use a version of this plasmid that is codon-optimized for E. coli
  11. NatB in pACYCduet vector (e.g., Addgene, catalog number: 53613 )
  12. Isopropyl β-D-1-thiogalactopyranoside (IPTG), > 99% (Ubiquitin-Proteasome Biotechnologies, catalog number: P1010-100 )
  13. Ampicillin sodium salt (Sigma-Aldrich, catalog number: A9518 )
  14. Chloramphenicol (Sigma-Aldrich, catalog number: C0378 )
  15. YT medium (2x) premixed powder (AppliChem, catalog number: A0981 )
  16. Magnesium sulfate heptahydrate p.a. (Merck, catalog number: 1058860500 )
  17. Glycerol bidistilled, ≥ 99.5% (VWR, catalog number: 24388 )
  18. Tris(hydroxymethyl)aminomethane, Trizma base p.a. (Sigma-Aldrich, catalog number: 93350 )
  19. Sodium chloride, p.a. (Carl Roth, catalog number: 3957 )
  20. Sodium dihydrogen phosphate monohydrate, p.a. (AppliChem, catalog number: 131965.1211 )
  21. α-Synuclein, N-terminally acetylated (from recombinant expression in E. coli [Johnson et al., 2010] and purified as described [Wördehoff et al., 2017])
    Note: We verify N-terminal acetylation by HPLC and mass spectrometry. Alternatively, acetic acid gel electrophoresis can be performed (Iyer et al., 2016). We have included a short description of our α-synuclein purification protocol in the procedure section of this protocol.
  22. Thioflavin T, ultrapure grade (AnaSpec, catalog number: AS-88306 )
  23. Sodium azide (purum p.a., Sigma-Aldrich, catalog number: 71290 )
  24. H2O (Milli-Q ≥ 18.2 MΩ resistivity)
  25. Glass beads, Ø 2.85-3.45 mm (Carl Roth, catalog number: A557.1 ), stored in 70% ethanol to ensure sterility
  26. Potassium dihydrogen phosphate (p.a., AppliChem, catalog number: A1043 )
  27. Dipotassium hydrogen phosphate, trihydrate (p.a., Merck, catalog number: 105099 )
  28. Potassium chloride (p.a., Carl Roth, catalog number: 6781.1 )
  29. Hydrochloric acid, p.a. ≥ 37% (Sigma-Aldrich, catalog number: 30721-M )
  30. Ammonium sulfate, puriss. p.a. ≥ 99% (Merck, catalog number: 31119-M )
  31. Modified 2YT medium (see Recipes)
  32. 10x Medium buffer, pH 7.2 (see Recipes)
  33. IEC buffer A (see Recipes)
  34. IEC buffer B (see Recipes)
  35. Saturated ammonium sulfate solution (see Recipes)
  36. SEC buffer (see Recipes)

Equipment

  1. Pipettes (e.g., Eppendorf, model: Research® Plus , catalog numbers: 3123000039, 3123000055, 3123000063)
  2. Sterile tweezers
  3. -80 °C freezer
  4. Fluorescence plate reader (e.g., BMG LABTECH, model: FLUOstar Omega ), equipped with monochromators or appropriate filters for the fluorophore Thioflavin T (e.g., BMG excitation filter: 448(nm)-10, BMG emission filter: 482(nm)-10)
    Note: The plate reader should be heatable to 37 °C.
  5. UV/VIS spectrophotometer (e.g., JASCO, model: V-650 )
  6. Heating Block (e.g., Grant Instruments, model: UBD2 )
  7. Magnetic stirrer (e.g., Cole-Parmer Instrument, Stuart, model: CB161 )
  8. Centrifuge for 50 ml Falcons (e.g., Eppendorf, model: 5804 R )
  9. ÄKTA Purifier (GE Healthcare)

Software

  1. Microsoft Excel
  2. AmyloFit (http://www.amylofit.ch.cam.ac.uk/)

Procedure

  1. Purification of acetylated α-synuclein
    Here we briefly describe how we purify our acetylated α-synuclein, as described in Wördehoff et al., 2017. As this protocol focusses on the aggregation of α-synuclein monitored by Thioflavin T fluorescence, the purification is described as concisely as possible.
    1. We coexpress codon-optimized α-synuclein in the pT7-7 vector together with the vector pNatB containing the N-terminal acetylation enzyme NatB from Schizosaccharomyzes pombe as described by Johnson et al., 2010.
    2. Expression is conducted in E. coli BL21DE3 grown in 2YT medium supplemented with 50 mM K/Na-phosphate buffer pH 7.2, 2 mM MgSO4 and 0.4% glycerol (v/v), with 100 μg/ml ampicillin and 35 μg/ml chloramphenicol as selection markers. We inoculate the (pre-)culture from a glycerol stock that has been prepared by overnight growth of a single colony in 2YT medium, mixed with 50% glycerol (sterile) and stored at -80 °C.
    3. After pregrowth to OD600 of 1-1.2, expression is induced by addition of 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) and incubated while shaking at 120 rpm for 4 h at 37 °C. We routinely prepare 500 ml culture, which gives us a final expression yield of ~25 mg acetylated α-synuclein.
    4. Cells are harvested by centrifugation at 6,000 x g for 15 min at 4 °C.
    5. An individual cell pellet (from 500 ml culture) is resuspended in 15 ml of pure H2O and frozen at -20 °C in 50 ml Falcon tubes.
    6. The Falcon tubes with the frozen cell suspension are transferred to a heat block preheated to 98 °C and incubated for 30 min to both lyse the cells and precipitate proteins by heat denaturation. α-Synuclein is thermostable and remains soluble.
    7. Cell lysate is cooled down on ice for 10 min, afterward cell debris and precipitated proteins are pelleted at 15,000 x g and 4 °C for 30 min.
    8. The supernatant is then filtered through a 0.2 μm filter into a new 50 ml tube.
    9. Saturated ammonium sulfate solution (~4.3 M at room temperature) is added in 1-ml steps to the supernatant to 50% saturation (1:1) while stirring on a magnetic stirrer, thereby precipitating α-synuclein, nucleic acids largely remain soluble.
    10. The turbid solution is then incubated on ice for 20 min and pelleted at 15,000 x g and 4 °C for 30 min.
    11. Supernatant is discarded and the pellet is resuspended in 50 ml of IEC buffer A (optional: dissolve in 5 ml IEC buffer A and dialyzed overnight against 1 L of IEC buffer A).
    12. The solution is then loaded onto a 5 ml HiTrap Q HP ion exchange chromatography column (GE Healthcare) connected to an ÄKTA purifier system (GE Healthcare), equilibrated in IEC buffer A.
    13. α-Synuclein elutes at around 250 mM NaCl on a 0-500 mM NaCl gradient to IEC buffer B, run over 100 ml at 2.5 ml/min.
    14. Fractions containing α-synuclein are combined, again precipitated by ammonium sulfate and pelleted as described above.
    15. The pellet is resuspended in 4 ml SEC buffer and finally purified in two runs by size exclusion chromatography (SEC) on a Superdex 75 16/60 column (120 ml column volume, GE Healthcare) at 1 ml/min in SEC buffer.
    16. After SEC, α-synuclein is sterile-filtered through a 0.2 μm filter and the concentration is measured on a spectrophotometer, using and extinction coefficient of 5,600 M-1 cm-1 at 275 nm.
    17. α-Synuclein is aliquoted, flash-frozen in liquid nitrogen and stored at -80 °C.

  2. General preparation
    1. Prepare stock solutions: 1 mM Thioflavin T in H2O, 10% (w/v) sodium azide in H2O. We use Thioflavin T solutions for a year; sodium azide solution is prepared fresh every month (both stored in the fridge at 4-8 °C).
    2. Buffers and protein solutions should be filter sterilized. As α-synuclein is sensitive to degradation, aliquots of α-synuclein should be stored at -80 °C and defrosted just before the assay.
    3. Calculate the amounts of reagents to pipette. For triplicate measurements, 400 μl sample per condition is needed (3x 120 μl, 40 μl reserve). In order to avoid that the ThT fluorescence saturates before the equilibrium of fibril formation is reached, a sufficiently high ThT concentration on the order of the applied protein concentration should be used. We typically use 10-20 μM ThT and 0.05% sodium azide (final concentrations).
    4. Include a negative control, which consists of buffer, salts, Thioflavin T and sodium azide.
    5. Preheat your plate reader to 37 °C. Aggregation assays can be performed at different temperatures, e.g., between 20 °C and 37 °C. However, we routinely use 37 °C as it is the human body temperature and α-synuclein aggregation is quite slow at lower temperatures.
    6. Prepare the plate: with sterile tweezers, take out as many glass beads as you need (out of the 70% ethanol solution), dry them shortly on a lint-less cloth and transfer them into the wells–choose glass beads of homogenous size. As the glass beads we use are slightly heterogeneous in terms of size and shape, we sort out glass beads with a small and large diameter, as well as glass beads that are not spherical by visual inspection. In general, the addition of glass beads is crucial to speed up aggregation and increase reproducibility of triplicates.
    7. We always leave the outer wells of the plate empty; they can give unexpected readings, so first well to use is B2, last well is G11.
    8. Optional: Pipette 120 μl water into the spaces between the wells and idle wells–this can be done to minimize possible evaporation from the sample wells.
    9. Prepare and label a sufficient amount of 1.5 ml protein low-bind reaction tubes and transfer tubes onto ice, do the subsequent pipetting steps on ice.

  3. Sample preparation
    1. Pipette the reagents, starting with the buffer, then sodium azide, Thioflavin T and lastly the protein solution into the 1.5 ml reaction tubes. As an example, this is the pipetting scheme for 400 μl of the 50 μM α-synuclein sample shown in Figure 1, assuming stock concentrations of 200 μM for α-synuclein, 1 mM Thioflavin T, and 10% sodium azide:
      1. 294 μl of SEC buffer
      2. 2 μl of 10% sodium azide
      3. 4 μl of 1 mM Thioflavin T
      4. 100 μl of α-synuclein (in SEC buffer) 
    2. Shortly (1 sec) vortex the samples to mix them and put them back on ice.
    3. Distribute 120 μl of sample per well.
    4. Seal plate with the sealing tape and transfer it to the plate reader.

  4. Plate reader settings
    1. We use a fluorescence plate reader (FLUOstar Omega by BMG LABTECH), assay temperature: 37 °C.
    2. Basic parameters: Microplate: Greiner 96 half area; Excitation filter: 448-10; Emission filter: 482-10; Gain: 750; Orbital Averaging–On: 3 mm, Bottom Optic, Settling time: 1 sec; Kinetic Window: 1,000 cycles, 12 flashes per well and cycle, cycle time: 900 sec.
    3. Layout: Mark the distribution of your samples.
    4. Shaking options: Orbital, 400 rpm, 30 sec before each cycle. Shaking is crucial to speed up aggregation and improve reproducibility of triplicates.
    5. Samples are run until fluorescence plateaus are reached.
    6. See also the appendix for the plate reader settings.

Data analysis

This section should guide the reader through the data analysis. We have included two exemplary aggregation assays of α-synuclein, the first showing the aggregation of α-synuclein at different concentrations (Figure 1), the second demonstrating the influence of an aggregation inhibitor (dityrosine-crosslinked α-synuclein), adapted from Wördehoff et al., 2017 (Figure 2).

  1. After the assay, we first calculate the mean of the negative control triplicates, i.e., the background fluorescence of Thioflavin T in our buffer, and subtract this mean background fluorescence from each of the sample triplicates at the given time points, e.g., in Microsoft Excel. In contrast to the negative control, the samples are not averaged and displayed as triplicates in the plot.
  2. The evaluation of the aggregation data can be done online with the AmyloFit Aggregation Fitter tool (http://www.amylofit.ch.cam.ac.uk/). AmyloFit can fit aggregation data and extract kinetic and mechanistic parameters of aggregation based on the latest mathematic models of amyloid aggregation (Meisl et al., 2016). If aggregation proceeds according to any of these models under the applied experimental conditions, and when fitted accurately and globally, parameters like primary/secondary nucleation rate constants and elongation rate constants can be extracted (Cohen et al., 2013).
  3. As an example, we monitored the aggregation of α-synuclein at four different concentrations and evaluated the data with AmyloFit (Figure 1).
  4. Data was exported to a tab-separated text file, with the first column being the time trace and the other ones being the fluorescence traces.
  5. As a first step, the raw data was plotted (see Figure 1A).
  6. The data was normalized within the Amylofit software (Figure 1B). This is done by choosing a data point (or a range of data points) in the fluorescence plateau at the end of the assay, which (or the average of which) is set to unity and all other values scaled accordingly. In the case of Figure 1B, the fluorescence intensities after 80 h (for 25 μM to 100 μM α-synuclein) or 90 h (for 10 μM α-synuclein) were used for normalization.
  7. The next step was plotting the aggregation half-times vs. the initial monomer concentration (m0) to extract the scaling exponent, which gives insight into which aggregation mechanisms are dominant (Figure 1C). Note that non-linear behavior in the double-logarithmic plot may be observed, pointing to a change in the dominant growth mechanisms with increasing protein concentration (Meisl et al., 2016). A selection flow chart for the dominant aggregation model to choose can be found in the supplementary information of the publication by Meisl et al., 2016.


    Figure 1. Aggregation kinetics of α-synuclein studied by ThT fluorescence. Four concentrations of α-synuclein (10 μM, 25 μM, 50 μM and 100 μM) aggregated in the presence of 10 μM Thioflavin T and 0.05% sodium azide in SEC buffer, measured in triplicates. The assay has been evaluated by the AmyloFit software (http://www.amylofit.ch.cam.ac.uk). A. Raw data, absolute ThT fluorescence readings of triplicate measurements of the four concentrations; B. Normalized aggregation data of the four concentrations; C. Double logarithmic half-time plot (log(m0): initial monomer concentration vs log(τ): aggregation half-time).

  8. The scaling exponent was -0.26, reflecting the low monomer concentration dependence of α-synuclein aggregation at neutral pH. This indicates that under these experimental conditions mechanisms are active that limit the monomer concentration dependence, such as saturating fibril elongation (Meisl et al., 2016; Buell et al., 2014), fibril fragmentation (Shvadchak et al., 2015), and nucleation at the air-water interface (Campioni et al., 2014), which may become saturated with protein (Pham et al., 2016).
  9. The assay can also be used to identify molecules that interfere with α-synuclein aggregation.
  10. As an example, we monitored the aggregation of α-synuclein in the absence and presence of dityrosine (DiY)-crosslinked α-synuclein (Wördehoff et al., 2017) (Figure 2A).
  11. It has to be noted that a reduced ThT fluorescence intensity does not necessarily mean that aggregation is inhibited, as inhibitors can also interact with ThT, obstruct ThT binding sites on fibrils and/or change the morphology of aggregates so that ThT can no longer bind (Jameson et al., 2012).
  12. To complement our ThT data, we therefore collected the samples with and without inhibitor after the assay and performed size exclusion chromatography to confirm that α-synuclein stays monomeric in the presence of the inhibitor (DiY α-synuclein) (Figure 2B).


    Figure 2. Aggregation kinetics of α-synuclein in presence of an inhibitor. 25 μM α-synuclein aggregated with (blue) and without (grey) 25 μM dityrosine (DiY)-crosslinked α-synuclein and 10 μM Thioflavin T and 0.05% sodium azide in SEC buffer, measured in triplicates. A. ThT fluorescence readings of triplicate measurements with and without inhibitor. B. Size exclusion chromatography of samples taken before and after the aggregation assay with and without inhibitor. Note that in presence of the inhibitor, the monomer peak after aggregation is equally high, whereas it decreased markedly without the inhibitor.

Recipes

  1. Modified 2YT medium (1 L)
    31 g YT medium (2x) premixed powder
    2 mM MgSO4
    0.4% Glycerol (v/v)
    Dissolve and mix with around 950 ml Milli-Q H2O and autoclave
    After autoclaving, mix 900 ml of the medium with 100 ml 10x medium buffer
    Store at room temperature
  2. 10x Medium buffer, pH 7.2 (1 l)
    Final concentration
    For 1 L
    360 mM K2HPO4
    82.16 g K2HPO4·3H2O
    140 mM NaH2PO4
    19.32 g NaH2PO4·H2O
    Dissolve in Milli-Q H2O and autoclave
    Store at room temperature
  3. IEC buffer A (50 mM Tris-HCl pH 8, for 500 ml)
    50 mM Tris(hydroxymethyl)aminomethane
    Dissolve 3.03 g Tris(hydroxymethyl)aminomethane in 400 ml Milli-Q H2O, titrate to pH 8 with HCl, adjust with Milli-Q H2O to 500 ml
    Store at room temperature
  4. IEC buffer B (50 mM Tris-HCl pH 8, 800 mM NaCl, for 500 ml)
    Final concentration
    For 500 ml
    50 mM Tris(hydroxymethyl)aminomethane
    3.03 g
    800 mM NaCl
    23.38 g NaCl
    Dissolve in 400 ml Milli-Q H2O, titrate to pH 8 with HCl, adjust with Milli-Q H2O to 500 ml
    Store at room temperature
  5. Saturated ammonium sulfate solution (250 ml)
    4.3 M Ammonium sulfate
    Dissolve 142.05 g (NH4)2SO4 in Milli-Q H2O and adjust the volume up to 250 ml with Milli-Q H2O and store at room temperature
  6. SEC buffer (1 L)
    Final concentration
    For 1 L
    20 mM K2HPO4
    4.56 g K2HPO4·3H2O
    5 mM KH2PO4
    0.68 g KH2PO4
    100 mM KCl
    7.46 g KCl
    Dissolve in Milli-Q H2O and filter sterilize
    The buffer is stored at room temperature

Acknowledgments

This method was adapted from Wördehoff et al. (2017). The plasmid pNatB (pACYCduet-naa20-naa25) was a gift from Dan Mulvihill (Addgene plasmid No. 53613). The project has received funding from the European Research Council under the European Union's Horizon 2020 research and innovation program, grant agreement No. 726368. The authors declare that no competing interests exist.

References

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简介

研究淀粉样蛋白如α-突触核蛋白体外聚集是一种方便和流行的工具,可以获得聚集的动力学见解以及研究因子(例如,聚集抑制剂) 影响它。 这些聚集测定通常利用荧光染料硫磺素T作为淀粉样蛋白原纤维形成的敏感荧光报告物,并且以基于读板器的形式进行,允许同时筛选多个样品和条件。 然而,由于原纤维成核的随机性质和调节因子的多样性,聚集测定通常倾向于较差的再现性。 在这里,我们提出了一个简单和可重复的协议,以研究基于读板器的测定中α-突触核蛋白的聚集。

【背景】内源性蛋白质与淀粉样原纤维的聚集是一种致病过程,与几种疾病相关,例如,神经退行性疾病如阿尔茨海默病(AD)或帕金森病(PD)以及全身性疾病如AL淀粉样变性( Knowles et al。,2014)。通过基于硫磺素T荧光的聚集测定,可以在基于板读者的装置中在体外中概括该过程,从而允许根据各种影响因素研究淀粉样蛋白的聚集动力学。

硫磺素T(ThT)是一种荧光染料,最初用于组织学样本中的淀粉样蛋白原纤维染色,于1959年由Vassar和Culling(Vassar和Culling,1959),其在体外检测和定量淀粉样纤维的应用
目前,硫代黄素T的聚集测定主要在荧光板读数器中进行,其中例如,96条件可以同时测试。由于原纤维成核的随机性质和影响蛋白质聚集的多种因素,这些测定法具有较差的重现性。因此,已经采用了增加ThT测定的再现性的策略,例如在测量期间使用井板的轨道摇动以及向孔中添加玻璃珠以改善混合(Giehm和Otzen,2010)。

在这里,我们描述了使用ThT进行α-突触核蛋白聚集测定的简单方案,其包括以下策略以提高再现性和方便性:


  1. 使用N-末端乙酰化的α-突触核蛋白,其是蛋白质的天然状态(Anderson 等人,,2006)。在ThT测定中,α-突触核蛋白的N-末端乙酰化也使聚集的再现性提高了一半(Iyer 等人,2016)。
  2. 在ThT荧光之前摇动30秒由于样品中原纤维的分布更均匀,包括优先在空气 - 水界面形成的聚集核,因此测量可以获得更可重复的荧光读数(Campioni et al。,2014)。 li>
  3. 每孔加入玻璃珠(Ø2.85-3.45mm),以改善样品的混合和均匀性(Giehm and Otzen,2010)。
  4. 我们使用半面积96 -well-plates(美国康宁),具有非结合表面。半面积孔可节省样品体积,因为每孔仅需100-120μl。非结合表面例如,防止淀粉样蛋白原纤维吸附到孔表面,这一过程可导致ThT测定中的异常读数(Murray 等人,,2013)。

  5. 关键字:淀粉样蛋白, 聚合, α-Synuclein, 硫磺素T检测, 帕金森病

    材料和试剂

    1. 50毫升Falcon离心管(TPP Techno Plastic Products,目录号:91050)
    2. 无菌注射器过滤器,Filtropur S,0.2μm(SARSTEDT,目录号:83.1826.001)
    3. Slide-A-Lyzer TM 透析盒10 kDA MW截止值,3-12 ml(Thermo Fisher Scientific,目录号:66810)
    4. HiTrap Q HP IEC色谱柱,5 ml(GE Healthcare,目录号:17115401)
    5. Superdex 75 16/60 SEC色谱柱,体积约120 ml(GE Healthcare,目录号:28989333)
    6. Protein LoBind Tubes 1.5 ml(Eppendorf,目录号:0030108116)
    7. 96孔板(康宁半面积,黑色和透明平底,非粘合表面)(康宁,目录号:3881)
    8. 密封胶带,透明聚烯烃(Thermo Fisher Scientific,目录号:232701)
    9. 电子。大肠杆菌 BL21DE3感受态细胞(例如,可从Merck获得,目录号:69450)
    10. pT7-7载体中的α-突触核蛋白(例如,Addgene,目录号:36046)
      我们使用该质粒的一个版本,该质粒针对 E进行了密码子优化。大肠杆菌
    11. pACYCduet载体中的NatB(例如,Addgene,目录号:53613)
    12. <异丙基β-D-1-硫代吡喃半乳糖苷(IPTG)> 99%(泛素 - 蛋白酶体生物技术,目录号:P1010-100)
    13. 氨苄青霉素钠盐(Sigma-Aldrich,目录号:A9518)
    14. 氯霉素(Sigma-Aldrich,目录号:C0378)
    15. YT中(2x)预混粉末(AppliChem,目录号:A0981)
    16. 硫酸镁七水合物 p.a。(默克,目录号:1058860500)
    17. 甘油双蒸馏,≥99.5%(VWR,目录号:24388)
    18. 三(羟甲基)氨基甲烷,Trizma碱 p.a。(Sigma-Aldrich,目录号:93350)
    19. 氯化钠, p.a。(Carl Roth,目录号:3957)
    20. 磷酸二氢钠一水合物, p.a。(AppliChem,目录号:131965.1211)
    21. α-突触核蛋白,N-末端乙酰化(来自大肠杆菌中的重组表达 [Johnson et al。,2010]并如所述纯化[Wördehoff et al。 ,2017])
      注意:我们通过HPLC和质谱验证了N-末端乙酰化。或者,可以进行乙酸凝胶电泳(Iyer等,2016)。我们在本协议的程序部分中简要介绍了我们的α-突触核蛋白纯化方案。
    22. 硫磺素T,超纯级(AnaSpec,目录号:AS-88306)
    23. 叠氮化钠( purum p.a。,Sigma-Aldrich,目录号:71290)
    24. H 2 O(Milli-Q≥18.2MΩ电阻率)
    25. 玻璃珠,直径2.85-3.45毫米(Carl Roth,目录号:A557.1),储存于70%乙醇中以确保无菌状态
    26. 磷酸二氢钾( p.a。,AppliChem,目录号:A1043)
    27. 磷酸氢二钾,三水合物( p.a。,Merck,目录号:105099)
    28. 氯化钾( p.a。,Carl Roth,目录号:6781.1)
    29. 盐酸, p.a。≥37%(Sigma-Aldrich,目录号:30721-M)
    30. 硫酸铵,puriss。 p.a。≥99%(默克,目录号:31119-M)
    31. 改良的2YT培养基(见食谱)
    32. 10x中等缓冲液,pH 7.2(见食谱)
    33. IEC缓冲区A(见食谱)
    34. IEC缓冲B(见食谱)
    35. 饱和硫酸铵溶液(见食谱)
    36. SEC缓冲区(见食谱)

    设备

    1. 移液器(例如,Eppendorf,型号:Research ® Plus,目录号:3123000039,3123000055,3123000063)
    2. 无菌镊子
    3. -80°C冰柜
    4. 荧光板读数器(例如,BMG LABTECH,型号:FLUOstar Omega),配有单色仪或适用于荧光团硫磺素T的滤光片(例如,BMG激发滤光片:448(nm) )-10,BMG发射滤光片:482(nm)-10)
      读板器应可加热至37°C
    5. UV / VIS分光光度计(例如,JASCO,型号:V-650)
    6. 加热块(例如,Grant Instruments,型号:UBD2)
    7. 磁力搅拌器(例如,Cole-Parmer仪器,Stuart,型号:CB161)
    8. 离心50 ml Falcons(例如,Eppendorf,型号:5804 R)
    9. ÄKTA净化器(GE Healthcare)

    软件

    1. Microsoft Excel
    2. AmyloFit( http://www.amylofit.ch.cam.ac.uk/ )

    程序

    1. 乙酰化α-突触核蛋白的纯化
      在这里,我们简要描述了我们如何纯化我们的乙酰化α-突触核蛋白,如Wördehoff et al。,2017所述。由于该协议主要关注由硫磺素T荧光监测的α-突触核蛋白的聚集,纯化是尽可能简洁地描述。
      1. 我们共同表达pT7-7载体中的密码子优化的α-突触核蛋白,以及来自 Schizosaccharomyzes pombe 的含有N-末端乙酰化酶NatB的载体pNatB,如Johnson 等所述。,2010。
      2. 表达在 E中进行。大肠杆菌BL21DE3在2YT培养基中生长,培养基中添加50 mM K /磷酸钠缓冲液pH 7.2,2 mM MgSO 4 和0.4%甘油(v / v),含100μg/ ml氨苄青霉素和35μg/ ml氯霉素作为选择标记。我们从甘油原液中接种(预)培养物,该甘油原液是通过在2YT培养基中过夜生长单个菌落,与50%甘油(无菌)混合制备的,并在-80℃下储存。
      3. 预生长至1-1.2的OD 600 后,通过加入1mM异丙基β-D-1-硫代吡喃半乳糖苷(IPTG)诱导表达,并在37℃下以120rpm振荡孵育4小时。 。我们经常准备500毫升培养物,这使我们的最终表达产量为~25毫克乙酰化α-突触核蛋白。
      4. 通过在4,000℃下以6,000 x g 离心15分钟收获细胞。
      5. 将单个细胞沉淀(来自500ml培养物)重悬于15ml纯H 2 O中,并在-20℃下在50ml Falcon管中冷冻。
      6. 将具有冷冻细胞悬浮液的Falcon管转移至预热至98℃的加热块中并孵育30分钟以裂解细胞并通过热变性沉淀蛋白质。 α-突触核蛋白是热稳定的并且保持可溶性。
      7. 将细胞裂解物在冰上冷却10分钟,然后将细胞碎片和沉淀的蛋白质在15,000 x g 和4℃下沉淀30分钟。
      8. 然后将上清液通过0.2μm过滤器过滤到新的50ml管中。
      9. 将饱和硫酸铵溶液(室温下~4.3M)以1ml步骤加入上清液至50%饱和度(1:1),同时在磁力搅拌器上搅拌,从而沉淀α-突触核蛋白,核酸基本上保持可溶。
      10. 然后将混浊的溶液在冰上孵育20分钟,并在15,000 x g 和4℃下沉淀30分钟。
      11. 弃去上清液,将沉淀重悬于50ml IEC缓冲液A中(任选:溶于5ml IEC缓冲液A中,对1L IEC缓冲液A透析过夜)。
      12. 然后将该溶液加载到连接到ÄKTA净化器系统(GE Healthcare)的5ml HiTrap Q HP离子交换色谱柱(GE Healthcare)上,在IEC缓冲液A中平衡。
      13. α-突触核蛋白在0-500mM NaCl梯度下以约250mM NaCl洗脱至IEC缓冲液B,以2.5ml / min运行超过100ml。
      14. 合并含有α-突触核蛋白的级分,再次通过硫酸铵沉淀并如上所述沉淀。
      15. 将沉淀重悬浮于4ml SEC缓冲液中,最后通过尺寸排阻色谱(SEC)在Superdex 75 16/60柱(120ml柱体积,GE Healthcare)上以1ml / min在SEC缓冲液中进行两次纯化。
      16. SEC后,通过0.2μm过滤器对α-突触核蛋白进行无菌过滤,并在分光光度计上测量浓度,使用和消光系数为5,600 M -1 cm -1 在275纳米。
      17. 将α-突触核蛋白等分,在液氮中快速冷冻并储存在-80℃。

    2. 一般准备
      1. 制备储备溶液:在H 2 O中的1mM硫磺素T,在H 2 O中的10%(w / v)叠氮化钠。我们使用硫磺素T溶液一年;每月新鲜制备叠氮化钠溶液(均在4-8°C的冰箱中保存)。
      2. 缓冲液和蛋白质溶液应过滤灭菌。由于α-突触核蛋白对降解敏感,α-突触核蛋白的等分试样应储存在-80°C并在试验前解冻。
      3. 计算移液器的试剂量。对于一式三份测量,每种条件需要400μl样品(3x120μl,40μl储备)。为了避免在达到原纤维形成平衡之前ThT荧光饱和,应该使用所施加的蛋白质浓度的足够高的ThT浓度。我们通常使用10-20μMThT和0.05%叠氮化钠(最终浓度)。
      4. 包括阴性对照,由缓冲液,盐,硫磺素T和叠氮化钠组成。
      5. 将读板器预热至37°C。聚集测定可以在不同的温度下进行,例如,例如,在20℃和37℃之间。然而,我们常规使用37°C,因为它是人体温度,并且在较低温度下α-突触核蛋白聚集非常慢。
      6. 准备好培养皿:用无菌镊子,取出所需数量的玻璃珠(从70%乙醇溶液中取出),在无绒布上快速干燥,然后将它们转移到孔中 - 选择同质大小的玻璃珠。由于我们使用的玻璃珠在尺寸和形状方面略微不均匀,我们挑选出具有小直径和大直径的玻璃珠,以及通过目视检查不是球形的玻璃珠。通常,添加玻璃珠对于加速聚集和提高一式三份的再现性至关重要。
      7. 我们总是将板的外孔留空;他们可以给出意想不到的读数,所以首先使用的是B2,最后好的是G11。
      8. 可选:移取120μl水进入井和空闲井之间的空间 - 这样做可以最大限度地减少样品井中可能的蒸发。
      9. 准备并标记足够量的1.5ml蛋白质低结合反应管并将管转移到冰上,在冰上进行随后的移液步骤。

    3. 样品制备
      1. 移取试剂,从缓冲液开始,然后用叠氮化钠,硫磺素T,最后将蛋白质溶液移入1.5ml反应管中。例如,这是图1中所示的400μl50μMα-突触核蛋白样品的移液方案,假设α-突触核蛋白,1 mM硫磺素T和10%叠氮化钠的原液浓度为200μM:
        1. 294μl的SEC缓冲液
        2. 2μl10%叠氮化钠
        3. 4μl的1mM硫磺素T
        4. 100μlα-突触核蛋白(在SEC缓冲液中)&nbsp;
      2. 不久(1秒)将样品涡旋混合,然后将它们放回冰上。
      3. 每孔分配120μl样品。
      4. 用密封胶带密封板并将其转移到读板器上。

    4. 读板设置
      1. 我们使用荧光板读数器(BMU LABTECH的FLUOstar Omega),测定温度:37°C。
      2. 基本参数:微孔板:Greiner 96半面积;励磁过滤器:448-10;发射滤光片:482-10;收益:750;轨道平均开:3毫米,底部光学,稳定时间:1秒;动力学窗口:1,000次循环,每孔12次闪烁和循环,循环时间:900秒。
      3. 布局:标记样品的分布。
      4. 摇动选项:轨道,400 rpm,每个循环前30秒。摇动对于加速聚合和提高一式三份的再现性至关重要。
      5. 样品运行直至达到荧光平台。
      6. 有关读板设置,另请参阅附录。

    数据分析

    本节应指导读者完成数据分析。我们已经包括两个示例性的α-突触核蛋白聚集测定,第一个显示不同浓度的α-突触核蛋白聚集(图1),第二个显示聚集抑制剂(二酪氨酸交联的α-突触核蛋白)的影响,改编自Wördehoff et al。,2017(图2)。

    1. 测定后,我们首先计算阴性对照一式三份的平均值,即,我们缓冲液中硫磺素T的背景荧光,并在给定时间减去每个样品一式三份的平均背景荧光在Microsoft Excel中指向例如。与阴性对照相反,样本不是平均值,并且在图中显示为三次。
    2. 可以使用AmyloFit Aggregation Fitter工具在线完成聚合数据的评估( http:// www .amylofit.ch.cam.ac.uk / )。 AmyloFit可以根据淀粉样蛋白聚集的最新数学模型拟合聚合数据并提取聚集的动力学和机械参数(Meisl et al。,2016)。如果在所应用的实验条件下根据任何这些模型进行聚集,并且当准确且全局拟合时,可以提取诸如一级/二级成核速率常数和伸长率常数的参数(Cohen 等人, 2013)。
    3. 例如,我们监测了四种不同浓度的α-突触核蛋白的聚集,并用AmyloFit评估了数据(图1)。
    4. 数据被导出到以制表符分隔的文本文件中,第一列是时间轨迹,另一列是荧光痕迹。
    5. 作为第一步,绘制原始数据(见图1A)。
    6. 数据在Amylofit软件中标准化(图1B)。这通过在测定结束时选择荧光平台中的数据点(或一系列数据点)来完成,其中(或其平均值)被设置为1并且所有其他值相应地缩放。在图1B的情况下,80小时后(对于25μM至100μMα-突触核蛋白)或90小时(对于10μMα-突触核蛋白)的荧光强度用于标准化。
    7. 下一步是绘制聚集半衰期与初始单体浓度( m0 )的关系,以提取比例指数,从而深入了解哪些聚合机制占主导地位(图1C)。注意,可以观察到双对数图中的非线性行为,指示随着蛋白质浓度增加主要生长机制的变化(Meisl 等人,2016)。选择主导聚合模型的选择流程图可以在Meisl 等人的出版物的补充信息中找到。,2016。


      图1.通过ThT荧光研究的α-突触核蛋白的聚集动力学在10μM硫磺素T存在下聚集的四种浓度的α-突触核蛋白(10μM,25μM,50μM和100μM)和在SEC缓冲液中的0.05%叠氮化钠,一式三份测量。该试验已通过AmyloFit软件( http://www.amylofit.ch.cam)进行评估。 ac.uk )。 A.原始数据,四种浓度的三次测量的绝对ThT荧光读数; B.四种浓度的归一化聚集数据; C.双对数半时间图(log( m0 ):初始单体浓度vs log(τ):聚合半衰期。

    8. 比例指数为-0.26,反映了在中性pH下α-突触核蛋白聚集的低单体浓度依赖性。这表明在这些实验条件下,机制是活性的,限制了单体浓度依赖性,例如饱和原纤维伸长率(Meisl 等。,2016; Buell et al。,2014 ),原纤维碎裂(Shvadchak et al。,2015),以及空气 - 水界面的成核(Campioni et al。,2014),可能会被蛋白质饱和(Pham et al。,2016)。
    9. 该测定还可用于鉴定干扰α-突触核蛋白聚集的分子。
    10. 例如,我们在不存在和存在二酪氨酸(DiY) - 交联的α-突触核蛋白的情况下监测α-突触核蛋白的聚集(Wördehoff等人,,2017)(图2A)。 >
    11. 必须注意的是,降低的ThT荧光强度并不一定意味着聚集被抑制,因为抑制剂也可以与ThT相互作用,阻碍原纤维上的ThT结合位点和/或改变聚集体的形态,使得ThT不再结合( Jameson et al。,2012)。
    12. 为了补充我们的ThT数据,我们因此在测定后收集含有和不含抑制剂的样品,并进行尺寸排阻色谱以证实α-突触核蛋白在抑制剂(DiYα-突触核蛋白)存在下保持单体(图2B)。 />

      图2.在抑制剂存在下α-突触核蛋白的聚集动力学。25μMα-突触核蛋白与(蓝色)和不含(灰色)25μM二酪氨酸(DiY) - 交联的α-突触核蛋白聚集在一起在SEC缓冲液中的μM硫磺素T和0.05%叠氮化钠,一式三份测量。 A.含有和不含抑制剂的三次测量的ThT荧光读数。 B.在有和没有抑制剂的聚集测定之前和之后采集的样品的尺寸排阻色谱。注意,在存在抑制剂的情况下,聚集后的单体峰同样高,而在没有抑制剂的情况下其显着降低。

    食谱

    1. 改良2YT培养基(1L)
      31克YT中(2x)预混粉末
      2 mM MgSO 4
      0.4%甘油(v / v)
      溶解并混合约950毫升Milli-Q H 2 O和高压灭菌器
      高压灭菌后,将900ml培养基与100ml 10x培养基缓冲液混合 在室温下储存
    2. 10x中等缓冲液,pH 7.2(1 l)
      最终集中
      1升
      360 mM K 2 HPO 4
      82.16 g K 2 HPO 4 ·3H 2 O
      140mM NaH 2 PO 4
      19.32 g NaH 2 PO 4 ·H 2 O
      溶于Milli-Q H 2 O和高压灭菌器中 在室温下储存
    3. IEC缓冲液A(50 mM Tris-HCl pH 8,500 ml)
      50mM三(羟甲基)氨基甲烷
      将3.03g三(羟甲基)氨基甲烷溶于400ml Milli-Q H 2 O中,用HCl滴定至pH8,用Milli-Q H 2 O调节至500ml
      在室温下储存
    4. IEC缓冲液B(50 mM Tris-HCl pH 8,800 mM NaCl,500 ml)
      最终集中
      适用于500毫升
      50mM三(羟甲基)氨基甲烷
      3.03克
      800 mM NaCl
      23.38克NaCl
      溶于400 ml Milli-Q H 2 O,用HCl滴定至pH 8,用Milli-Q H 2 O调节至500 ml
      在室温下储存
    5. 饱和硫酸铵溶液(250毫升)
      4.3 M硫酸铵
      在Milli-Q H 2 O中溶解142.05 g(NH 4 ) 2 SO 4 并调节体积用Milli-Q H 2 O至250ml并在室温下储存
    6. SEC缓冲液(1 L)
      最终集中
      1升
      20mM K 2 HPO 4
      4.56克K 2 HPO 4 ·3H 2 O
      5mM KH 2 PO 4
      0.68克KH 2 PO 4
      100 mM KCl
      7.46克KCl
      溶于Milli-Q H 2 O并过滤灭菌
      缓冲液储存在室温下

    致谢

    该方法改编自Wördehoff等人(2017)。质粒pNatB(pACYCduet-naa20-naa25)是来自Dan Mulvihill的礼物(Addgene质粒No.53613)。该项目已获得欧洲研究理事会根据欧盟“地平线2020”研究和创新计划,第726368号赠款协议提供的资金。作者声明不存在竞争利益。

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引用:Wördehoff, M. M. and Hoyer, W. (2018). α-Synuclein Aggregation Monitored by Thioflavin T Fluorescence Assay. Bio-protocol 8(14): e2941. DOI: 10.21769/BioProtoc.2941.
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Aderemi A
Yokohama City Univ
Hi,

Are these glass beads re-usable? If yes, how are they processed or washed to enable re-use? Thanks. Remi.
2018/11/20 23:08:38 回复
Michael Wördehoff
Physikalische Biologie, Heinrich-Heine-Universität, Germany, Germany,

Hey Remi,
The glass beads are (theoretically) reusable after thorough cleaning. However, we only use them once and omit the cleaning procedure, as the beads are quite inexpensive.
Best, Michael

2018/11/21 4:15:35 回复