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Aug 2020

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Yeast Lipid Extraction and Analysis by HPTLC
酵母脂质的HPTLC提取及分析   

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Abstract

The diversity of lipid structures, properties, and combinations in biological tissues makes their extraction and analysis an experimental challenge. Accordingly, even for one of the simplest single-cellular fungi, the budding yeast (Saccharomyces cerevisiae), numerous extraction and analysis protocols have been developed to separate and quantitate the different molecular lipid species. Among them, most are quite sophisticated and tricky to follow. Herein, we describe a yeast total lipids extraction procedure with a relatively good yield, which is appropriate for subsequent thin-layer chromatography (TLC) or liquid chromatography-mass (LC-MS) analysis. We then discuss the most widely used solvent systems to separate yeast phospholipids and neutral lipids by TLC. Finally, we describe an easy and rapid method for silica gel staining by a Coomassie Brilliant Blue-methanol mixture. The stained lipid species can then be quantitated using imaging software such as ImageJ. Overall, the methods described in this protocol are time-saving and novice-friendly.

Keywords: Lipid extraction (脂质提取), TLC (TLC), Lipid staining (脂质染色), Phospholipid (磷脂), Neutral lipid (中性脂质), Budding yeast (出芽酵母)

Background

Lipid research has gained great momentum in recent years, stimulated in part by the interest in lipid-associated disorders in humans, in part by aspirations of biofuel and biopharmaceutical production. Lipids play vital roles in many biological processes, including the formation of bio-membranes, the storage of energy, and numerous signal transduction processes. The budding yeast, Saccharomyces cerevisiae, has been an excellent model organism for studying the physiological and pathophysiological roles of lipids at the cellular level (Popa et al., 2016). Primary lipid metabolism is highly conserved between yeast and humans in both the basic biochemical pathways and the regulatory circuitries that link lipid metabolism to energy homeostasis, cell growth, and development. One advantage of using yeast in lipid-related research is that the pathways and molecular compositions are often less complicated than those in mammals, facilitating their scientific investigation. In yeast, PA (phosphatidate acid), PC (phosphatidylcholine), PE (phosphatidylethanolamine), PI (phosphatidylinositol), PS (phosphatidylserine), DAG (diacylycerols), and their respective lyso-derivatives are the most abundant membrane lipid classes, while PG (phosphatidylglycerol) and CL (cardiolipin) are less abundant. TAG (triacylglycerol) and SE (sterol ester) act as storage lipids. The most abundant yeast sphingolipids consist of IPC (inositolphosphoceramide), MIPC (mannose inositolphosphoceramide), and M(IP)2C (mannose diinositolphosphoceramide) (Klug and Daum, 2014).


Lipid extraction procedures described in the literature generally share common principles but vary in processing steps. Among these, the most cited Folch method uses CHCl3/CH3OH (2:1 v/v) as the solvent system (Folch et al., 1957). Alternate proportions of CHCl3/CH3OH or replacement of CHCl3 with 2-propanol or hexane have also been utilized (Fuchs et al., 2015; Knittelfelder and Kohlwein, 2017a and 2017b). In this protocol, we use CHCl3/CH3OH (2:1 v/v) as the solvent for lipid extraction and break yeast cells with glass beads in a mechanical bead mill. We repeat the extraction five times to ensure efficient lipid extraction.


Silica gel is the most wildly used stationary phase for chromatographic lipid separation. Impregnated with different substances, modified silica gel can be used to separate various lipid classes. Among the most popular modifications, silver nitrate impregnation has better potential for separating glycerolipids containing unsaturated fatty acyl chains (Dobson et al., 1995); boric acid is primarily used to separate the various isomers of DAG or PL (Ando et al., 2000). Like in other chromatographic techniques, TLC can be classified as “normal” or “reversed” phase based on the polarities of their mobile and stationary phases. Normal phase (polar stationary phase and non-polar mobile phase) chromatography is the standard method to separate lipids of interest that may have different polarities caused by a variation in their head groups (Fuchs et al., 2011). Typically, the size of gel particles for TLC is 10-50 μm. In contrast, the size of the high-performance thin-layer chromatography (HPTLC) gel particles is about 5 μm with narrow distributions (Fuchs et al., 2011), which results in higher separation quality and smaller sample quantity. It is noteworthy that bio-lipids are very complex molecules, and no single separation method is sufficient to separate all lipid species. With the stationary phase chosen, the separating efficiency of TLC or HPTLC mainly relies on the mobile phase. A 0.4% (NH4)SO4-impregnated silica gel plate together with chloroform-methanol-acetic acid-acetone-water (40:25:7:4:2 v/v/v/v/v) as the mobile phase can be used to separate phospholipids, lysophospholipids, and SM (sphingomyelin) (Wang and Gustafson, 1992). A chloroform-methanol-water mixture (35:15:2 or 50:40:10 v/v/v) is generally used for the separation of SM; a different proportion, chloroform-methanol-water mixture (65:25:4 v/v/v), can be used to separate phospholipids (Knittelfelder and Kohlwein, 2017a and 2017b). A mobile phase consisting of hexane-diethyl ether-acetic acid (70/30/1 v/v/v) is initially used to separate cholesterol isomers; it can also be used to separate neutral lipids such as TAGs, DAGs, and MAGs (monoacylglycerols). Alternatively, neutral lipids can be separated by TLC silica gel 60 plates and petroleum ether-diethyl ether-acetic acid (32:8:0.8 v/v/v/) mixture as the mobile phase (Knittelfelder and Kohlwein, 2017a and 2017b). Overall, it is a trial-and-error process to choose a proper stationary phase and mobile phase based on the characteristics of individual lipid species. In the present protocol, a silica gel 60 plate is used as the stationary phase, and a hexane-diethyl ether-acetic acid (70/30/1 v/v/v) mixture and a chloroform-methanol-water mixture (65:25:4 v/v/v) are used as the mobile phase to separate neutral lipids and phospholipids, respectively. Note that the mobile phase for chromatographic lipid separation is not to be confused with the solvent system for lipid extraction, which is discussed in the preceding paragraph.


After separation, the resulting positions of different lipid species on TLC plates need to be visualized, usually by color reactions with chemical spray reagents. An iodine vapor bath is one of the most widely used methods. The brown iodine color will disappear spontaneously once the plates are removed from the iodine vapor (Palumbo and Zullo, 1987). It has been reported that iodine can be difficult to remove from highly unsaturated lipids, but in our experience, the brown color fades away too quickly before follow-up steps can be properly performed. Alternative spray reagents include 2,7-dichlorofluorescein, rhodamine 6 G, and primuline. Together with iodine, they belong to non-destructive reagents, with the modification of lipid structures being reversible (Fuchs et al., 2011). 0.2% amino black 10 B in 1 M NaCl and 3.2% H2SO4, 0.5% MnCl2 in 50% ethanol are two destructive reagents with high sensitivity (Fuchs et al., 2011 and 2015; Knittelfelder and Kohlwein, 2017a and 2017b). In this protocol, we use Coomassie Brilliant Blue R-250, a non-destructive reagent first used to stain lipids in 1984 (Nakamura and Handa, 1984). In our experience, the detection sensitivity and durability of Coomassie Brilliant Blue R-250 are better than those of iodine vapor. Conveniently, the staining steps only take 30 min.


Materials and Reagents

  1. Pipette tips (Thermo Fisher Scientific, different sizes and types)

  2. Centrifuge tube, 5-ml round-bottomed (Sangon Biotech, catalog number: F610888)

  3. Cryogenic microtubes, 1.5-ml deep cap, conical-bottomed, sterile (Sangon Biotech, catalog number: F600154)

  4. Gel blotting paper (Sangon Biotech, catalog number: F513323)

  5. Yeast extracts (OXCID, catalog number: LP0021)

  6. Peptone (BD, catalog number: 211677)

  7. D-glucose (Sangon Biotech, catalog number: A501991)

  8. Yeast nitrogen base without amino acids and ammonium sulfate (Sangon Biotech, BBI, catalog number: A600505)

  9. Chloroform (General-Reagent, catalog number: G75915B)

  10. Methanol (General-Reagent, catalog number: G75851B)

  11. Coomassie Brilliant Blue R-250 (Sangon Biotech, catalog number: CB0037)

  12. Hexane (General-Reagent, catalog number: G14153D)

  13. Diethyl ether (HUSHI, catalog number: 10009318)

  14. Acetic acid (General-Reagent, catalog number: G73562B)

  15. Lipid standard: 18:1 DG (1,2-dioleoyl-sn-glycerol) 2 mg/ml chloroform (Avanti, catalog number: 800811C)

  16. Lipid standard: Egg PC (L-alpha-phosphatidylcholine) 10 mg/ml chloroform (Avanti, catalog number: 840051C)

  17. TLC Silica gel 60 (25 aluminium sheets 20 × 20cm; Merk Millipore, catalog number: 105553)

  18. Glass thin-layer chromatography (TLC) developing chambers (100 × 100, generic)

  19. Glass beads (40 or 50 mesh, generic)

Equipment

  1. Pipettes (Thermo Fisher Scientific, different types)

  2. Shaking incubator (CRYSTAL, model: IS-RDH1)

  3. Spectrophotometer (Shanghai Jing-Hua, model: 722S)

  4. Centrifuge (Thermo, model: SORVALL ST16R)

  5. Freeze-dryer/lyophilizer (Labconco, model: Freezone 6 Plus)

  6. Analytical scale (Shanghai Heng-Ping, model: FA2104)

  7. Drying oven (BOXUN, model: GZX-9146MBE)

  8. Bead mill (Shanghai Jing-Xin, model: Tissue lyser-96)

  9. Thermomixer (ALLSHENG, model: MSC-100)

  10. TLC developing chamber with cover (generic)

  11. Gel imaging system (Bio-Rad, model: Gel Doc 721BR05189)

Software

  1. ImageJ (https://imagej.net/Downloads)

Procedure

  1. Cell growth and harvest

    1. Grow yeast cells in 100 ml YPD medium (1% yeast extract, 2% peptone, 2% glucose) overnight in a 30°C shaking incubator (250 rpm) until the optical density (OD600) of the culture is about 0.7-1.0.

    2. Harvest the cells by centrifugation, 10,000 × g for 5 min at room temperature. Remove the supernatant completely and discard.

    3. Freeze-dry the cells in a freeze-dryer and store at -80°C.


  2. Total lipid extraction

    1. For each freeze-dried sample, transfer 0.25 mg into a 1.5-ml cryogenic microtube.

    2. Add 200 μl washed glass beads to each sample tube.

    3. Add 500 μl chloroform-methanol (2:1, v/v) to each sample tube. Screw on the Teflon-sealed cap tightly.

    4. Place the sample tubes in a bead mill and perform mechanical shearing at maximum speed for 5 min at room temperature. Repeat the mechanical lysis twice; rearrange tube locations in-between to avoid uneven cell lysis (Step 1 in Figure 1).

    5. Centrifuge the cell lysates at room temperature, 10,000 × g for 5 min. After centrifugation, cell debris will be sandwiched between the supernatant and the glass beads (Step 2 in Figure 1). Transfer the supernatant to new tubes for collection (Step 3 in Figure 1).

    6. Repeat the chloroform-methanol extraction procedure (Steps 2-6 in Figure 1) five times.

    7. Merge the supernatant collections of each sample in a 5-ml tube and evaporate on a compact thermomixer at 60°C overnight with the lid off. This step needs to be performed in a fume hood. After evaporation, membrane-like substances will be left at the bottom of the tubes.



      Figure 1. Schematic depiction of the lipid extraction procedure


  3. Lipid analysis by TLC

    1. Dissolve the lipid extracts in 100-500 μl chloroform.

    2. Prepare the solvent mixtures:

      1. For separation of phospholipids, prepare a chloroform-methanol-H2O (65:25:4, v/v/v) mixture (Knittelfelder and Kohlwein, 2017a and 2017b).

      2. For separation of neutral lipids, prepare a hexane-diethyl ether-acetic acid (70:30:1 v/v/v) mixture (Fuchs et al., 2015).

    3. Add the mobile phase to glass TLC developing chambers. Saturate the chamber atmosphere with folded blotting paper for more than 30 min.

    4. Activate the silica gel 60 plates at 100°C in an oven for 30 min. Draw a fine spotting line with a pencil 2 cm from the bottom of the TLC plate.

    5. Spot 5-20 μl dissolved samples on the activated silica gel plates carefully with 10-μl pipette tips. Keep the spacing between these spots larger than 0.5 cm. In addition, spot 0.2-1 μg lipid standards (DAG or PC) to distinguish the complex bands of each sample and to monitor the developing and staining steps.

    6. Develop each plate in a solvent chamber until the solvent front reaches the top of the plate. To maintain a solvent-saturated atmosphere in the chamber, do not open the lid while the plate is developing.

    7. Dry the plates in a fume hood for 20 min at room temperature.

    8. Stain the dried plates with a 0.03% Coomassie Brillant Blue R-250 solution containing 20% methanol for 15 min, and subsequently de-stain in 20% methanol for 10 min (Nakamura and Handa, 1984).

    9. Dry the plates under room temperature for 1 h and take photos by a gel imaging system (Figure 2).

    10. Analyze the grayscale of each lipid band using ImageJ (Steps: Open an image file in ImageJ – go to Analyze – gels – Select First Line – Select Next Line – Wand (tracing) Tool – Plot Lanes).



      Figure 2. Examplary HPTLC separation of yeast lipids. A. Neutral lipids: the solvent system was hexane-diethyl ether-acetic acid (70:30:1 v/v/v). DAG, diacylglycerol; TAG, triacylglycerol; SE, steryl esters. B. Polar lipids: the solvent system was chloroform-methanol-H2O (65:25:4, v/v/v). FA, fatty acids; NL, neutral lipids; PE, phosphatidylethanolamine; CL, cardiolipin; PC, phosphatidylcholine; PS, phosphatidylserine. X, unidentified. Lipids were stained with Coomassie Brillant Blue R-250.

Acknowledgments

The authors would like to thank Dr. Zhou Peng and Prof. Xiaoling Miao (School of Life Sciences and Biotechnology, Shanghai Jiao Tong University) for their kind advice regarding experimental procedures. This work was supported by the National Natural Science Foundation of China (grants 91957104, 31671431, and 91754110). This protocol is adapted from Li et al. (2020) and works cited therein.

Competing interests

The authors declare no competing financial interests

References

  1. Ando, Y., Satake, M., and Takahashi., Y. (2000). Reinvestigation of positional distribution of fatty acids in docosahexaenoic acid-rich fish oil triacyl-sn-glycerols. Lipids 35(5): 579-582.
  2. Dobson, G., Christie, W.W., and Nikolova-Damyanova., B. (1995). Silver ion chromatography of lipids and fatty acids. J Chromatogr B Biomed Appl 671(1-2): 197-222.
  3. Folch, J., Lees, M. and Sloane Stanley, G. H. (1957). A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem 226(1): 497-509.
  4. Fuchs, B., Süss, R., Teuber, K., Eibisch, M. and Schiller, J. (2011). Lipid analysis by thin-layer chromatography--a review of the current state. J Chromatogr A 1218 (19): 2754-2774.
  5. Fuchs, B., Popkova, Y., Süß, R. and Schiller, J. (2015). Separation of (Phospho)Lipids by Thin-Layer Chromatography. In: Instrumental Thin-Layer Chromatography. 2015: 375-405.
  6. Klug, L. and Daum, G. (2014). Yeast lipid metabolism at a glance. FEMS Yeast Res 14(3): 369-388.
  7. Knittelfelder, O. L. and Kohlwein. S. D. (2017a). Lipid Extraction from Yeast CellsCold Spring Harb Protoc 2017(5): pdb prot085449.
  8. Knittelfelder, O. L. and Kohlwein, S. D. (2017b). Thin-Layer Chromatography to Separate Phospholipids and Neutral Lipids from Yeast. Cold Spring Harb Protoc 2017(5).
  9. Nakamura, K. and Handa, S. (1984). Coomassie brilliant blue staining of lipids on thin-layer plates. Anal Biochem 142(2): 406-410.
  10. Popa C, Coll NS, Valls M and Sessa G. (2016). Yeast as a Heterologous Model System to Uncover Type III Effector Function. PLoS Pathog 12(2): e1005360.
  11. Palumbo, G. and Zullo, F. (1987). The use of iodine staining for the quantitative analysis of lipids separated by thin layer chromatography. Lipids 22(3): 201-205.
  12. Wang, W.Q. and Gustafson. A. (1992). One-dimensional thin-layer chromatographic separation of phospholipids and lysophospholipids from tissue lipid extracts. J Chromatogr 581(1): 139-142.

简介

[摘要]生物组织中脂质结构、性质和组合的多样性使其提取和分析成为实验挑战。因此,最简单的单一的,甚至对于一个-细胞真菌,芽殖酵母(酿酒酵母),大量的提取和分析的协议已被开发,以分离和孔定量泰特不同分子脂质物种。其中,大多数都非常复杂且难以遵循。本文,我们描述了酵母的总脂质的提取方法,用一个相对LY良好产率,这是应用程序 适合随后的薄层色谱 (TLC) 或液相色谱-质量 (LC-MS) 分析。然后,我们讨论了通过 TLC 分离酵母磷脂和中性脂质的最广泛使用的溶剂系统。最后,我们描述一个简单的和快速的通过为硅胶染色法考马斯乙rilliant乙略-甲醇混合物。将染色的脂质物种C的然后是孔定量达ED使用成像软件如ImageJ的。总体而言, 本协议中描述的方法既省时又适合新手。

[背景]唇ID的研究已经取得了巨大的momentu近岁男,刺激部分通过在人体脂质相关疾病的兴趣,部分吸小号的生物燃料和生物制药生产。血脂起到至关重要的作用在许多生物过程,包括形成的生物-膜小号,能量的储存,以及众多的信号转导过程。芽殖酵母,酿酒酵母,得到了极好的模式生物用于学习的在细胞水平的脂质的生理学和病理生理学作用(波帕等人,2016)。主要脂质代谢高度酵母和人之间保守的小号在基本生化途径和监管电路系统都该链接脂质代谢能量平衡,细胞生长和发育。的一个优势利用酵母脂质-相关的研究是,途径和分子组成的往往低于那些在哺乳动物中,复杂的有利于自己的科学考察。在酵母中,PA(磷脂酸),PC(磷脂酰胆碱),PE(磷脂酰乙醇胺),PI(磷脂酰肌醇),PS(磷脂酰丝氨酸),DAG(diacylycerols ),以及它们各自的溶血-衍生物是最丰富的膜脂类,而PG (磷脂酰甘油)和 CL(心磷脂)含量较少。TAG(甘油三酯)和 SE(甾醇酯)充当储存脂质。最丰富的酵母神经鞘脂类由IPC(的我nositolphosphoceramide ),MIPC(米annose inositolphosphoceramide ),和M(IP)2 C(米annose diinositolphosphoceramide )(克鲁格和Daum的,201 4)。

大号IPID提取程序描述于文献中通常具有共同的原则,但变种ý在处理步骤。其中,引用最多的Folch方法使用CHCl 3 /CH 3 OH (2:1 v/v) 作为溶剂系统(Folch等,1957)。甲lternate比例三氯甲烷的3 / CH 3 OH或更换的氯仿3用2-丙醇或己烷也已被利用(福克斯等人,2015; Knittelfelder和Kohlwein ,2017年一和2017b )。在该协议中,我们使用CHCl 3 /CH 3 OH (2:1 v/v) 作为脂质提取溶剂,并在机械珠磨机中用玻璃珠破碎酵母细胞。我们重复提取五次以确保有效的脂质提取。

硅胶是色谱脂质分离中使用最广泛的固定相。浸渍differen吨物质,改性的二氧化硅凝胶可用于分离各种脂质类。在最流行的改进中,硝酸银浸渍具有更好的分离含有不饱和脂肪酰基链的甘油脂的潜力(Dobson等,1995);硼酸主要用于小号eparate的各个DAG或PL(安藤的异构体等。,2000)。与其他色谱技术一样,TLC 可以根据其流动相和固定相的极性分为“正相”或“反相”。正相(极性固定相和非极性流动相)色谱法是标准的方法,以单独的脂质感兴趣的是可具有引起不同极性一个变化在他们的头部基团(福克斯等人,2011)。通常,TLC 的凝胶颗粒大小为 10 – 50 μm 。与此相反,大小高性能薄层色谱法(HPTLC )凝胶颗粒为约5微米与窄分布(福克斯等人,2011) ,其结果小号在更高的分离质量和更小的样品量。这是笔记值得Ÿ是生物脂质是非常复杂的分子,并没有单一的分离方法足以分离所有脂质物质。与固定相选择的,TLC或HPTLC的分离效率主要依赖于所述流动相。阿0.4%(NH 4 )SO 4 -浸渍的硅胶板一起用氯仿-甲醇-乙酸-丙酮-水(40:25:7:4:2 V / V / V / V / V)作为流动相可用于分离磷脂小号,溶血磷脂,和SM(小号phingomyelin)(王和古斯塔夫森,1992)。A C hlor oform -甲醇-水混合物(35:15:2或50:40:10 V / V / V)是通常用于SM的分离; 不同比例的氯仿-甲醇-水混合物(65:25:4 v/v/v) 可用于分离磷脂(Knittelfelder和Kohlwein ,2017a和2017b )。阿米obile相由己烷-乙醚-乙酸(70/30/1 v / v / v)的最初用于单独的胆固醇异构体; 它也可以用来分离中性脂质小号如标记,的DAG ,并在MAG (单酰基甘油)。或者,可以通过 TLC 硅胶 60 板和石油醚-二乙醚-乙酸 (32:8:0.8 v/v/v/) 混合物作为流动相分离中性脂质(Knittelfelder和Kohlwein ,2017a和2017b )。总的来说,这是一个TR IA升-和错误的过程来选择一个基于个别脂质类物质的特性适当的固定相和流动相。在本协议中,一个硅胶60板我š使用作为固定相,和己烷-乙醚-乙酸(70/30/1 v / v / v)的混合物和一个氯仿-甲醇-水混合物(65 :25:4 v / v / v)被使用为t他移动相来分离中性立的PID和分别磷脂,。注意的是,流动相为色谱类脂物的分离是不与用于脂质提取,这是在前面的段落中所讨论的溶剂系统相混淆。

甲压脚提升的分离,所得到的位置需要被可视化,通常是不同的脂质种类的在TLC板上通过显色反应与化学喷雾试剂。一个I odine VAP或浴是最广泛的一个使用的方法。一旦将板从碘蒸气中取出,棕色碘就会自然消失(Palumbo和Zullo ,1987 年)。据报道,碘可能很难从高度不饱和脂质清除,b在我们的经验掉得太快,可以正确地进行后续步骤之前UT,棕色的颜色变淡。替代喷雾试剂包括 2,7-二氯荧光素、罗丹明 6 G 和樱草碱。连同碘,它们属于非-破坏性试剂,与脂质的修饰结构小号是可逆的(福克斯等人,2011)。0.2%氨基黑10中的B的1M NaCl和3.2%H 2 SO 4 ,0.5%的MnCl 2在50%乙醇是具有两个破坏性试剂高灵敏度(福克斯等人,2011和2015; Knittelfelder和Kohlwein ,2017A和2017b )。在这个协议中,我们使用考马斯乙rilliant乙略R-250 ,一个非-破坏性试剂首先用于染色脂质在1984年(中村和半田,1984)。根据我们的经验,的检测灵敏度和耐久性考马斯乙rilliant乙略R-250是优于那些的碘蒸气。方便的是,染色步骤只需30 分钟。

关键字:脂质提取, TLC, 脂质染色, 磷脂, 中性脂质, 出芽酵母

材料和试剂
移液器吸头(Thermo Fisher Scientific,不同尺寸和类型)
离心管中,5 -毫升园-底部ED (生工生物,目录号:F610888)
低温微管小号,1.5 -毫升深帽,圆锥形-底部ED ,无菌(生工生物,目录号:F600154)
凝胶印迹婷纸(生工生物,目录号:F513323)
酵母提取物(OXCID,目录号:LP0021)
蛋白胨(BD,目录号:211677)
D-克lucose(生工生物,目录号:A501991)
的酵母氮基质无氨基酸和硫酸铵(生工生物,BBI,目录号:A600505)
氯仿(常规- - [R eagent,目录号:G75915B)
甲醇(常规- - [R eagent,目录号:G75851B)
考马斯乙rilliant乙略R-250(生工生物,目录号:CB0037)
己烷(常规- - [R eagent,目录号:G14153D)
乙醚(HUSHI,目录号:10009318)
乙酸(常规- - [R eagent,目录号:G73562B)
大号IPID标准:18:1 DG(1,2-二油酰基-sn-甘油)2毫克/毫升Ç hloroform(阿凡提,目录号:800811C)
大号IPID标准:蛋PC(L-α- p hosphatidylcholine)10毫克/毫升Ç hloroform(阿凡提,目录号:840051C)
TLC硅胶60(25个铝片20 × 20cm ; Merk Millipore,目录号:105553)
玻璃薄层色谱 (TLC) 显影室(100 × 100,通用)
玻璃b EADS(40或50目,通用)




设备


移液器(Thermo Fisher Scientific,不同类型)
SHAK荷兰国际集团培养箱(CRYSTAL,米Odel等:IS-RDH1)
分光光度计(上海景华,米奥德尔:722S)
离心机(热,米Odel等:SORVALL ST16R)
冷冻-干燥机/冻干机(LABCONCO ,米Odel等:自由区6加)
一个nalytical规模(上海恒平,米奥德尔:FA2104)
干燥炉(博讯,米Odel等:GZX-9146MBE)
珠磨机(上海荆-X中,米Odel等:组织lyser-96)
Thermomixer中(ALLSHENG,米Odel等:MSC-100)
带盖TLC显影室(通用)
凝胶成像系统(Bio-Rad公司,米Odel等:Gel Doc凝胶721BR05189)


软件


ImageJ ( https://imagej.net/Downloads )


程序


细胞生长和收获
生长酵母在10 ST细胞0毫升YPD培养基(1%酵母提取物,2%蛋白胨,2%葡萄糖)中过夜以3 0 ℃下SHAK荷兰国际集团培养箱(250rpm下)直到光密度(OD 600 )的培养物是大约 0.7 - 1.0。
通过离心收获细胞,10 ,000 ×克,在室温下5分钟。完全去除上清液并丢弃。
冷冻干燥的细胞在一个冷冻-博士揭掉并储存在-80℃。


总脂质提取
对于每一个冷冻干燥的样品,转移0.25毫克到1.5 -毫升低温微管中。
向每个样品管中加入200 μl洗涤过的玻璃珠。
向每个样品管中加入 500 μl氯仿-甲醇 (2:1, v/v) 。螺钉上的特氟隆密封帽紧紧。
放置在样品管中一个珠密耳升和在最大速度下5分钟进行机械剪切在室温下。重复机械裂解两次冰;重新排列中间的试管位置以避免细胞裂解不均匀(图 1 中的步骤1)。 
离心的细胞溶胞产物在室温下,万×克5分钟。离心后,细胞碎片将被夹在之间的上清液和玻璃珠(步骤2在图1中)。转移的上清液到新桶Ë小号收集(步骤3在图1中)。
重复氯仿-甲醇萃取程序(步骤小号2 - 6图1中)的五倍。
合并上清液集合的ë ACH样品中的5 -毫升管和上紧凑恒温在60过夜蒸发℃下与所述盖关闭。此步骤需要在通风橱中进行。甲压脚提升蒸发,膜样物质将留在该管的底部。


C:\Users\Madandan\Desktop\2003753--1802 谢志平\Figs tif\图1.tif


图1.小号化学ATIC的升的描绘IPID提取过程


TLC 脂质分析
将脂质提取物溶解在100 - 500 μl氯仿中。
准备溶剂混合物:
为了分离磷脂,制备氯仿-甲醇-H 2 O(65:25:4,v/v/v)混合物(Knittelfelder和Ko hlwein ,2017a和2017b )。
为了分离中性脂质,制备己烷-乙醚-乙酸 (70:30:1 v/v/v) 混合物(Fuchs等人,2015 年)。
将流动相添加到玻璃 TLC 显影室中。小号aturate所述室气氛与折叠印迹廷纸超过30分钟。
将硅胶 60 板在 100°C的烘箱中激活30 分钟。用铅笔在距 TLC 板底部 2 cm 处画一条细线。
点5 - 20个微升上样溶解在活化硅胶板小心地用10 -微升移液管尖端。保持这些点之间的间距大于 0.5 厘米。另外,发现0.2 - 1微克脂质标准(DAG或PC)来区分每个样品的复杂带和到监控显影和染色小号TEP秒。
在溶剂室中展开每个板,直到溶剂前沿到达板的顶部。为了保持在室中的溶剂的饱和气氛中,而不用打开盖板被显影。
干燥该板在通风橱中对20分钟在室温下。
染色的干燥板用0.03%考马斯亮含有20%甲醇15蓝R-250溶液分钟,和随后的去污渍在20%甲醇,10分钟(中村和半田,1984)。
干燥室温下将板1个小时并拍照通过凝胶成像小号ystem(图URE 2 )。
分析灰度每种脂质频带的使用的ImageJ (š依照步骤:ø笔一个N个图像文件中的ImageJ -去分析-凝胶-选择第一行-选择下一行-棒(跟踪)Ť OOL -剧情泳道)。


C:\Users\Madandan\Desktop\2003753--1802 谢志平\Figs tif\图2.tif


˚F igure 2.为例进制ħ PTLC分离酵母脂质。一个。中性脂质:溶剂系统是己烷-二乙醚-乙酸(70:30:1 v/v/v) 。DAG,甘油二酯;TAG,三酰甘油;SE,甾醇酯。乙。极性脂质:溶剂系统是氯仿-甲醇-H 2 O (65:25:4, v/v/v) 。FA,脂肪酸;NL,中性脂质;PE,磷脂酰乙醇胺;CL,心磷脂;PC,磷脂酰胆碱;PS,磷脂酰丝氨酸。X,身份不明。大号ipids染色用考马斯马斯亮蓝R-250 。


致谢


作者要感谢周鹏博士和苗晓玲教授(上海交通大学生命科学与生物技术学院)对实验程序的友好建议。这项工作得到了中国国家自然科学基金 (grant s 91957104、31671431 和 91754 110) 的支持。该协议改编自Li 等人。, BMC Biol 18, 107 (2020)和其中引用的著作。


利益争夺


作者声明没有竞争性经济利益


参考


安藤,Y.,佐竹,M. ,和Takahashi。, Y. (2000)。重新研究富含二十二碳六烯酸的鱼油三酰基-sn-甘油中脂肪酸的位置分布。脂质35(5):579 5 82。
多布森,G.,克里斯蒂,WW ,和Nikolova-Damyanova 。, B. (1995)。脂质和脂肪酸的银离子色谱。J Chromatogr B Biomed Appl 671(1-2):197-222。
Folch , J., Lees, M. 和 Sloane Stanley , G. H. (1957)。一种从动物组织中分离和纯化总脂质的简单方法。J Biol Chem 226(1): 497-509。
福克斯,B.,苏斯,- [R 。,特伯,K 。,艾比施,M 。和席勒,J. (2011 年)。通过薄层色谱法进行脂质分析——对当前状态的回顾。Ĵ Chromatogr甲1218 (19):2754- 27 74。
福克斯,B.,Popkova ,Y.,s ^ USS ,- [R 。和席勒,J. (2015 年)。通过薄层色谱法分离(磷)脂质。在:仪器薄层色谱法。2015 年:375-405。
Klug, L. 和Daum , G. (2014)。酵母脂质代谢一目了然。FEMS酵母RES 14(3):369- 3 88。
Knittelfelder , O. L. 和Kohlwein 。S. D. (2017 a )。从酵母细胞中提取脂质。Cold Spring Harb Protoc 2017(5): pdb prot085449。
Knittelfelder , O. L. 和Kohlwein , S. D. (201 7 b ) 。从酵母中分离磷脂和中性脂质的薄层色谱法。Cold Spring Harb Protoc 2017(5)。
Nakamura, K. 和Handa , S. (1984)。薄层板上脂质的考马斯亮蓝染色。肛门生物化学142(2):406- 4 10。
Popa C、Col NS、Valls M和Sessa G. (2016 年)。酵母作为异源模型系统来揭示 III 型效应子功能。PLoS 病原体12(2):e1005360。
Palumbo, G. 和Zullo , F. (1987)。使用碘染色对薄层色谱分离的脂质进行定量分析。脂质22(3):201- 20 5。
王、WQ 和古斯塔夫森。A. (1992)。从组织脂质提取物中对磷脂和溶血磷脂进行一维薄层色谱分离。ĴChromatogr 581(1):139- 1 42。
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引用:Li, D., Zhang, Z., He, C. and Xie, Z. (2021). Yeast Lipid Extraction and Analysis by HPTLC. Bio-protocol 11(13): e4081. DOI: 10.21769/BioProtoc.4081.
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