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Dye Release Experiments with Dextran Loaded Vesicles
采用载有右旋糖酐的囊泡进行染色释放试验   

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Abstract

Dye release experiments are a widely used method to assess the interactions between membrane-active molecules and lipid membranes. Of particular interest is the ability to assess the degree of the lipid bilayer perturbation by simultaneously encapsulating dye of different sizes, such as dextrans grafted with a chromophore. In this assay, dextran linked to rhodamine or fluorescein are both encapsulated in lipid vesicles to allow quantifying the leakage of each dextran individually from a single sample. For instance, the size evaluation of the lipid pore formed by an antimicrobial peptide has been recently achieved using this protocol (Sani et al., 2013).

Keywords: Model membranes(模型膜), Phospholipid bilayers(磷脂双分子层), Dye leakage(染料渗漏), Fluorescence assay(荧光测定法), Antimicrobial peptides(抗菌肽)

Materials and Reagents

  1. Rhodamine-dextran (RD) of different molecular weights [for instance 40 kDa RD molecular weight (RD-40)]
  2. Fluorescein-dextran (FD) of different molecular weights [for instance 4.4 kDa FD (FD-4)]
  3. Tris.HCl
  4. NaCl
  5. MQ-water
  6. Triton-X100
  7. Peptide of interest e.g. Maculatin 1.1 (Sani et al., 2013)
  8. Lipids of interest e.g. 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), 1',3'-bis[1,2-dioleoyl-sn-glycero-3-phospho]-sn-glycerol (TOCL) (Sani et al., 2013)

Equipment

  1. Polycarbonate membrane filters (200 nm diameter)
  2. Extruder (for instance Avanti Mini-extruder, Alabaster)
  3. Bench centrifuge
  4. Spectrofluorimeter and quartz cuvette

Procedure

  1. Stock preparation
    1. Prepare a Tris.HCl + X NaCl buffer (buffer A) and a Tris.HCl + (X-Dextran concentration) NaCl buffer (buffer B) at desired pH, with X being the NaCl concentration.
    2. Prepare a peptide stock solution (The concentration will depend on the peptide solubility and the required lipid to peptide molar ratio under investigation.) in buffer A. Glass vials are recommended.
    3. Prepare a stock solution of equimolar RD and FD in buffer B.
    4. Co-solubilize lipids in chloroform/methanol (3: 1 v/v), remove the organic solvents under vacuum using a rotary evaporator. Rehydrate the lipids in Milli-Q water and lyophilize.
    5. Resuspend the require mass of fluffy lipid powder with the dextran stock solution. A 15 mM lipid stock solution usually allows practical dilution to reach required lipid to peptide molar ratio, but will be dependent on the peptide stock concentration. Perform 3-5 freeze-thaw cycles to homogenise the liposome dispersion, usually without a time delay between cycles, by dipping the solution in liquid nitrogen and then melting above the lipid fluid phase transition for ~ 5 min.
    6. Extrude 10 times through an Extruder using 0.2 mm pore size polycarbonate filters to produce large unilamellar vesicles (LUV) of 200 nm diameter. Extrusion must be performed above the gel-to-fluid lipid phase transition to avoid vesicle aggregation or demixing of heterogeneous lipid compositions, if used.
    7. Remove un-encapsulated dye from LUV dispersions by centrifuging three times at 22,000 x g for 30 min at 25 °C. After each centrifugation, remove the supernatant and replace with an equal volume of fresh buffer B solution and mixed gently.
    8. Determine the lipid concentration of washed LUV dispersions in triplicate using the phosphorus assay of Sani et al. (2013).

  2. Sample preparation
    1. Equilibrate the LUV dispersion and peptide solution at the desired temperature.
    2. Depending on fluorimeter performance, prepare an adequate dextran-encapsulated solution, ~ 200 μM gives a high signal in our experience.
    3. Add the require amount of peptide to obtain the lipid to peptide molar ratio (L/P) of interest. For reproducibility and statistical analysis, it is best to prepare the sample in triplicate.
    4. Produce negative control by adding buffer B instead of the peptide solution. A non-membrane active peptide or protein, such as bovine serum albumim, can also be used.
    5. Produce positive control by adding 0.5% Triton X-100 (Tx).
    6. Incubate the samples for 30 min at the desired temperature.
    7. Centrifuge the samples at 22,000 x g for 30 min.
    8. Recover the supernatant, with care to leave the pellet unperturbed. Usually, 500 μl sample are prepared and 300 μl recovered for analysis.
    9. Transfer the supernatant to a quartz cuvette.

  3. Fluorescence measurement
    1. Record the fluorescence emission of RD (then FD) using an excitation wavelength set at 550 nm (then 480 nm). Fluorescence emission is recorded from 560 to 650 nm using adequate integration time and PMT voltage. Triplicate the measurements.
    2. The amount of fluorescence is determined by integrating the area under the emission curve. The % release of each sample is calculated by using the following equation: where, I is the fluorescence intensity of peptide treated vesicle supernatant, I0 obtained from the peptide-free sample (negative control) supernatant, and Imax from the Tx treated supernatant (positive control).

Acknowledgments

None.

References

  1. Anderson, R. L. and Davis, S. (1982). An organic phosphorus assay which avoids the use of hazardous perchloric acid. Clinica Chimica Acta 121(1): 111-116.
  2. Sani, M. A., Whitwell, T. C., Gehman, J. D., Robins-Browne, R. M., Pantarat, N., Attard, T. J., Reynolds, E. C., O'Brien-Simpson, N. M. and Separovic, F. (2013). Maculatin 1.1 disrupts Staphylococcus aureus lipid membranes via a pore mechanism. Antimicrob Agents Chemother 57(8): 3593-3600.

简介

染料释放实验是一种广泛使用的方法来评估膜 - 活性分子和脂质膜之间的相互作用。 特别感兴趣的是通过同时包封不同尺寸的染料(例如用发色团接枝的葡聚糖)来评估脂质双层扰动的程度的能力。 在该测定中,连接到罗丹明或荧光素的葡聚糖都封装在脂质囊泡中,以允许从单个样品单独地量化每个葡聚糖的渗漏。 例如,最近使用该方案获得了由抗微生物肽形成的脂质孔的尺寸评价(Sani等人,2013)。

关键字:模型膜, 磷脂双分子层, 染料渗漏, 荧光测定法, 抗菌肽

材料和试剂

  1. 不同分子量的罗丹明葡聚糖(RD)[例如40kDa RD分子量(RD-40)]
  2. 不同分子量的荧光素 - 葡聚糖(FD)[例如4.4kDa FD(FD-4)]
  3. Tris HCl
  4. NaCl
  5. MQ水
  6. Triton-X100
  7. 感兴趣的肽例如 Maculatin 1.1(Sani et al。,2013)
  8. 感兴趣的脂质例如1-棕榈酰-2-油酰-sn-甘油基-3-胆碱磷酸(POPC),1-棕榈酰-2-油酰-sn-甘油基-3-磷酸乙醇胺(POPE),1 - 棕榈酰-2-油酰-sn-甘油基-3-磷酸甘油(POPG),1',3'-双[1,2-二油酰基-sn-甘油-3-磷酸] > sn - 甘油(TOCL)(Sani et al。,2013)

设备

  1. 聚碳酸酯膜过滤器(直径200nm)
  2. 挤出机(例如Avanti小型挤出机,Alabaster)
  3. 台式离心机
  4. 分光荧光计和石英比色皿

程序

  1. 库存准备
    1. 制备Tris缓冲盐水+ X NaCl缓冲液(缓冲液A)和Tris缓冲液。 (X-葡聚糖浓度)NaCl缓冲液(缓冲液B)在所需pH下 是NaCl浓度。
    2. 制备肽储备溶液   (该浓度将取决于肽的溶解度 在研究中所需的脂质与肽的摩尔比)。   建议使用玻璃小瓶。
    3. 在缓冲液B中制备等摩尔RD和FD的储备溶液
    4. 共溶解脂质在氯仿/甲醇(3:1 v/v)中,除去 有机溶剂在真空下使用旋转蒸发器。 再水合 脂质在Milli-Q水中并冻干
    5. 重新挂起require 质量的蓬松脂质粉末与葡聚糖储备溶液。 A 15mM 脂质储备溶液通常允许实际稀释达到所需  脂质与肽的摩尔比,但将取决于肽 库存浓度。进行3-5次冻融循环以均质化 脂质体分散,通常在周期之间没有时间延迟 将溶液浸入液氮中,然后在脂质上熔化  流体相变〜5分钟。
    6. 挤出10次 使用0.2mm孔径聚碳酸酯过滤器的挤出机来生产 大单层囊泡(LUV)。挤出必须 在凝胶 - 液体脂质相转变之上进行,以避免囊泡  聚集或分离异质脂质组合物(如果使用)
    7. 通过离心三次从LUV分散体中除去未包封的染料 次,在22,000×g下在25℃下30分钟。每次离心后, 除去上清液并用等体积的新鲜缓冲液B替换  溶液并轻轻混合。
    8. 使用Sani等人的磷测定法一式三份确定洗涤的LUV分散体的脂质浓度。 (2013年)。

  2. 样品制备
    1. 在所需的温度下平衡LUV分散体和肽溶液
    2. 根据荧光计的性能,准备足够的 葡聚糖封装溶液,〜200μM在我们的中给出高信号 经验。
    3. 添加所需量的肽以获得 脂质与肽的摩尔比(L/P)。 重复性和 统计分析,最好一式三份准备样品
    4. 通过添加缓冲液B而不是肽来产生阴性对照 解。 非膜活性肽或蛋白质,例如牛血清   albumim,也可以使用。
    5. 通过加入0.5%Triton X-100(Tx)产生阳性对照。
    6. 在所需温度下孵育样品30分钟。
    7. 将样品以22,000×g离心30分钟。
    8. 回收上清液,小心保持沉淀不受干扰。 通常,制备500μl样品,并回收300μl用于分析。
    9. 将上清液转移到石英比色皿。

  3. 荧光测量
    1. 使用激发记录RD(然后FD)的荧光发射 波长设定在550nm(然后480nm)。 荧光发射 使用足够的积分时间和PMT从560至650nm记录 电压。 将测量值重复三次。
    2. 大量的 通过积分发射下的面积来确定荧光 曲线。 通过使用以下来计算每个样品的%释放   方程:其中,I是肽处理的荧光强度 囊泡上清液,从无肽样品获得的I阴性(阴性) 对照)上清液,和来自Tx处理的上清液的I max   控制)。

致谢

没有。

参考文献

  1. Anderson,R.L。和Davis,S。(1982)。 有机磷测定,避免使用危险的高氯酸。 Clinica Chimica Acta 121(1):111-116。
  2. Sani,M.A.,Whitwell,T.C.,Gehman,J.D.,Robins-Browne,R.M.,Pantarat,N.,Attard,T.J.,Reynolds,E.C.,O'Brien-Simpson,N.M.和Separovic, Maculatin 1.1通过孔隙机制破坏了金黄色葡萄球菌脂质膜。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Sani, M., O’Brien-Simpson, N. M. and Separovic, F. (2014). Dye Release Experiments with Dextran Loaded Vesicles. Bio-protocol 4(14): e1190. DOI: 10.21769/BioProtoc.1190.
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