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A Technique for the Measurement of in vitro Phospholipid Synthesis via Radioactive Labeling
采用放射性标记的体外磷脂合成测量法   

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参见作者原研究论文

本实验方案简略版
Biochemical Journal
Aug 2014

Abstract

This is an assay designed to examine the radioactive phosphorous incorporation when the molecule is being synthesized, which means that only de novo synthesized phospholipids can be detected. Thus, with this technique it is possible to detect in vitro phospholipid synthesis under different required experimental conditions respect to controls (Guido and Caputto, 1990; Ferrero et al., 2014). There are different types of lipids. Among them we can find phospholipids, which contain glycerol esterified with two fatty acyl chains and a phosphate group that can also be bound to an organic molecule that acts as “hydrophilic head”, as shown in Figure 1 for the case of phosphatidylcholine. This structure confers amphipathic properties to lipid molecules that allow them to form lipid bilayers, making phospholipids the main components of biological membranes.


Figure 1. Representation of phospholipid structure. Extracted from: http://bio1151.nicerweb.com/Locked/media/ch05/phospholipid.html

Keywords: Phospholipids Synthesis (磷脂的合成), Radioactive (放射性), In vitro (体外), ATP-P32 (atp-p32), Labeling (标记)

Materials and Reagents

  1. PYREX® 5 ml Rimless Kahn Culture Tubes (12 x 75 mm) (Corning, catalog number: 9820-12 )
  2. Scintillation vials (Sigma-Aldrich, catalog number: Z190527 )
  3. The enzymes and substrates are obtained as protein homogenates from homogenized cells or tissue (see Step 1 of the Procedure)
  4. Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad Laboratories, AbD Serotec®, catalog number: 5000006 )
  5. HEPES (Sigma-Aldrich, catalog number: H3375 )
  6. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
  7. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333 )
  8. D-(+)-Glucose (Sigma-Aldrich, catalog number: G8270 )
  9. Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M8266 )
  10. 32P]ATP (PerkinElmer, catalog number: BLU002001MC )
  11. Chloroform (Sigma-Aldrich, catalog number: C2432 )
  12. Methanol (Sigma-Aldrich, catalog number: 34860 )
  13. Trichloroacetic acid (TCA) (Sigma-Aldrich, catalog number: T6399 )
  14. Phosphotungstic acid (PTA) (Sigma-Aldrich, catalog number: P4006 )
  15. Non-aqueous liquid scintillation cocktail (PerkinElmer, catalog number: 1200-434 )
  16. Buffer HEPES (see Recipes)
  17. Reaction buffer (see Recipes)
  18. TCA-PTA 10-1 (% w/v) (see Recipes)
  19. TCA-PTA 5-0.5 (% w/v) (see Recipes)
  20. Carrier brain homogenate (see Recipes)

Equipment

  1. Tip sonicator (Branson Sonic Power Company, model: Sonifier B-12 ) or ULTRA-TURRAX®
  2. Spectrophotometer (Wavelength to be used: 595 nm) (Shimadzu Scientific Instruments, model: BioSpec-mini )
  3. Vortex (IKA® VORTEX 3) (Sigma-Aldrich, catalog number: Z654779 )
  4. Pipettes (PIPETMAN® L Starter Kit) (Gilson, catalog number: F167350 )
  5. Centrifuge (Cavourargetina, model: VT-3216Dx24 )
  6. Water thermostatic bath (Vicking, model: Masson D )
  7. Scintillation counter (WinSpectral, Wallac, PerkinElmer)
  8. Laboratory prepared with all the required equipment for safe receiving, storage, manipulation and waste processing of radioactive material, according to laws governing in each country
  9. Geiger counter (Ludlum Measurements, model: 3 survey meter )

Procedure

Note: To determine under which conditions lipid synthesis is linear with time, prepare different tubes and carry out the reaction at 37 °C for different times. If you are evaluating the capacity of modifying lipid synthesis of a given protein, you must also determine under which conditions lipid synthesis is linear with the concentration of your protein of interest.
The person that will carry on the experiments must have strict training with radioactive working, according to rules and governing laws.

  1. Incorporation of radioactive 32P into cell/tissue homogenates
    1. Grow the desired cell line with the required culture medium on a 10 cm dish until reaching 90% confluence.
      Note: At this point, if you need your cells to be cultured under special conditions or subjected to a particular treatment, you should adequate the protocol to fit your needs.
    2. Harvest cells with 300 µl of milliQ water or use a homogenizer or ULTRA-TURRAX® if the sample is obtained from a tissue, to obtain the protein homogenates according to experimental requirements.
    3. Sonicate on ice and measure total protein concentration using Bradford method or a similar one.
    4. Maintain samples on ice all the time.
    5. Prepare the reaction tubes. Each of them must contain a final volume of 80 µl, with 1x reaction buffer, 3 μCi of [γ32P] ATP and a protein concentration of 1.25 mg/ml. Consider that the samples must be measured at least in triplicate. For this, first aliquot in a Kahn tube the amount of protein homogenate necessary to reach the required protein concentration. Then, add the adequate volumes of the reaction buffer and the [γ32P] ATP to reach the concentrations mentioned above. As normally there are several conditions to be measured, and taking into account that they must be performed at least in triplicate, we find it convenient to prepare a master mix for all the tubes to avoid pipetting mistakes. For this, first aliquot the protein homogenate in the different tubes, then prepare a master mix with the adequate amounts of reaction buffer and [γ32P] ATP and aliquot it in the tubes. Finally, add enough milliQ water to reach the final volume.
      Note: To simplify, it would help to normalize the protein concentration of all the samples so the volume of the master mix to be added is the same for all the tubes. Also, prepare a volume a bit larger of the master mix than the theoretical requirement, to compensate small volume losses or pipette miscalibrations. Adjust the final volume with milliQ water.
    6. Vortex for 10 sec.
    7. Carry out the reaction at 37 °C for 60 min.
      Note: At this time you can stop the reaction by freezing or you can continue with the purification steps. If you stop the reaction by freezing, the sample must be put in a bunker to avoid irradiation, according to working conditions with radioactive material.
    8. Add 80 μl of TCA-PTA 10-1 (% w/v) to precipitate lipids and proteins. At this point, you should start seeing the imminent formation of a white precipitate. Vortex for 10 sec and centrifuge for 10 min at 3,500 rpm.

  2. Lipids precipitation and purification
    1. Discard the supernatant, which contains the free [γ32P] ATP that has not been incorporated, and vortex until the pellet is disaggregated.
    2. Add 1 ml of TCA-PTA 5-0.5 (% w/v) and vortex again for 10 sec. Centrifuge and repeat the pellet wash four times. Discard the supernatant after each round.
      Note: If the amount of precipitate is too small, you can add 5 µl of brain homogenate, which will function as a carrier (see Figure 2).


      Figure 2. Example of the expected amount of precipitate (right tube) after adding the carrier brain homogenate. The tube on the left shows a precipitate considered too small.

    3. Carry on a final wash with milliQ water, to favor lipid dissolution in the organic solvent (step B12).
    4. After centrifugation, discard the water supernatant of the last wash and vortex until the pellet is disaggregated.
    5. Add 1.5 ml of chloroform:methanol 2:1 and vortex for 10 sec. The chloroform dissolves the lipids and the methanol maintains the proteins precipitated.
    6. Centrifuge 10 min at 3,500 rpm to allow protein precipitation and separation from soluble lipids.
    7. Transfer 1.3 ml of the supernatant containing the marked lipids to a vial and dry in a water bath at 90 °C for 3 h or at room temperature overnight. When transferring the supernatant, be careful and do not pull precipitated proteins that may be marked with 32P.
      Caution: As this step involves the transfer of a chloroform containing solution, glass pipettes previously equilibrated with chloroform should be used to avoid sample loss.
      Note: There is a loss of 0.2 ml when transferring the supernatant to avoid disturbing the pellet.

  3. Measurement of 32P content in purified lipids
    1. Add 1 ml of non-aqueous scintillation cocktail, tap and vortex for 10 sec.
    2. Determine the levels of incorporated 32P in the lipids using a liquid scintillation counter. The reactions must be performed in triplicate, as minimum.

  4. Data analysis
    1. The results can be expressed as absolute values (as shown in Figure 3) or as relative values respect to a control condition taken as 100% of incorporation.

Representative data

Figure 3 is a representative example of data that indicates the type of results expected. In this case, the experimental condition measures the amount of phospholipid synthesis in the presence of c-Fos protein, its phosphorylated version or its mutants. In this regard, results are represented as amount of phospholipid labeling (Ferrero et al., 2012).


Figure 3. c-Fos phosphorylated by c-Src does not activate phospholipid synthesis. The capacity to activate phospholipid synthesis of recombinant c-Fos, c-Fos phosphorylated by purified c-Src (P-c-Fos), the phosphomimetic Y10/30E mutant of c-Fos and the non-phosphorylatable mutant of c-Fos Y10/30F was examined as described previously (Gil et al., 2004). Incubations were for 60 min at 37 °C. 32P-phospholipid quantification was performed as described previously (Guido and Caputto, 1990). Results expressed as c.p.m. of 32P incorporated into phospholipids/mg of protein are the mean±s.d. of three experiments performed in triplicate; *P<0.002 with respect to control (buffer) as determined by One Way ANOVA analysis. Y10/30F not incubated with c-Src and c-Src incubated without any added substrates were run as controls. Note that the presence of c-Src in the assays did not modify phospholipid synthesis.

Notes

If you decide to freeze after carrying out the reaction in step A6, don’t add the TCA-PTA 10-1 (% w/v) solution as the homogenate might agglomerate and precipitate together with non incorporated [γ32P] ATP molecules, which can lead to erroneous measurements.

Recipes

  1. Buffer HEPES (2 M, pH 7.5)
    Dissolve 476.6 g HEPES in 800 ml of H2O
    Adjust pH to 7.5 with the appropriate volume of concentrated NaOH
    Add H2O to a final volume of 1 L
  2. Reaction buffer (20x, 20 ml)

    Reagent
    Quantity
    Concentration
    NaCl
    3.2726 g
    2,800 mM
    KCl
    0.1491 g
    100 mM
    MgCl2
    0.019 g
    10 mM
    Glucose
    0.4036 g
    112 mM
    HEPES buffer (2 M, pH 7.5)
    12.8 ml

    Add milliQ water to a final volume of 20 ml
    The buffer is stable for six months if stored at -20 °C.
  3. TCA-PTA 10-1 (% w/v)
    Weight 10 grams of TCA and 1 gram of PTA and add milliQ water to a final volume of 100 ml
  4. TCA-PTA 5-0.5 (% w/v)
    Aliquot 50 ml of the TCA-PTA 10-1 and add milliQ water to a final volume of 100 ml
  5. Carrier brain homogenate
    Euthanize an adult Wistar rat and excise the brain
    Weight and prepare an homogenate of 1 g tissue per ml of milliQ water

Acknowledgments

We thank Dr. Beatriz L. Caputto for helpful discussions to adapt and modify the protocol form her previous work (Guido and Caputto, 1990). We also thank CONICET and FONCYT for funding.

References

  1. Guido, M. E. and Caputto, B. L. (1990). Labeling of retina and optic tectum phospholipids in chickens exposed to light or dark. J Neurochem 55(6): 1855-1860.
  2. Ferrero, G. O., Renner, M. L., Gil, G. A., Rodriguez-Berdini, L. and Caputto, B. L. (2014). c-Fos-activated synthesis of nuclear phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] promotes global transcriptional changes. Biochem J 461(3): 521-530.
  3. Ferrero, G. O., Velazquez, F. N. and Caputto, B. L. (2012). The kinase c-Src and the phosphatase TC45 coordinately regulate c-Fos tyrosine phosphorylation and c-Fos phospholipid synthesis activation capacity. Oncogene 31(28): 3381-3391.

简介

这是设计用于当合成分子时检查放射性磷掺入的测定,这意味着可以检测到只有新合成的磷脂。因此,利用该技术,可以在相对于对照的不同所需实验条件下检测体外磷脂合成(Guido和Caputto,1990; Ferrero等人,2014) 。有不同类型的脂质。其中我们可以找到磷脂,其含有用两个脂肪酰基链酯化的甘油和磷酸基团,磷酸基团也可以结合到充当"亲水头部"的有机分子,如图1所示的磷脂酰胆碱的情况。这种结构赋予脂质分子两亲性质,使它们形成脂质双层,使磷脂成为生物膜的主要成分。


图1。磷脂结构的代表。摘录自: http://bio1151.nicerweb .com/Locked/media/ch05/phospholipid.html

关键字:磷脂的合成, 放射性, 体外, atp-p32, 标记

材料和试剂

  1. 5ml Rimless Kahn培养管(12×75mm)(Corning,目录号:9820-12)
  2. 闪烁瓶(Sigma-Aldrich,目录号:Z190527)
  3. 酶和底物作为来自匀浆细胞或组织的蛋白质匀浆获得(参见步骤的步骤1)
  4. Bio-Rad蛋白测定染料试剂浓缩物(Bio-Rad Laboratories,AbD Serotec ,目录号:5000006)
  5. HEPES(Sigma-Aldrich,目录号:H3375)
  6. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S7653)
  7. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9333)
  8. D - (+) - 葡萄糖(Sigma-Aldrich,目录号:G8270)
  9. 氯化镁(MgCl 2)(Sigma-Aldrich,目录号:M8266)
  10. [γsup 32 P] ATP(PerkinElmer,目录号:BLU002001MC)
  11. 氯仿(Sigma-Aldrich,目录号:C2432)
  12. 甲醇(Sigma-Aldrich,目录号:34860)
  13. 三氯乙酸(TCA)(Sigma-Aldrich,目录号:T6399)
  14. 磷钨酸(PTA)(Sigma-Aldrich,目录号:P4006)
  15. 非水液体闪烁鸡尾酒(PerkinElmer,目录号:1200-434)
  16. 缓冲HEPES(参见配方)
  17. 反应缓冲液(参见配方)
  18. TCA-PTA 10-1(%w/v)(参见配方)
  19. TCA-PTA 5-0.5(%w/v)(参见配方)
  20. 载体脑匀浆(见配方)

设备

  1. Tip Sonicator(Branson Sonic Power Company,型号:Sonifier B-12)或ULTRA-TURRAX
  2. 分光光度计(使用波长:595nm)(Shimadzu Scientific Instruments,型号:BioSpec-mini)
  3. Vortex(IKA VORTEX 3)(Sigma-Aldrich,目录号:Z654779)
  4. 移液管(PIPETMAN L Starter Kit)(Gilson,目录号:F167350)
  5. 离心机(Cavourargetina,型号:VT-3216Dx24)
  6. 水恒温槽(Vicking,型号:Masson D)
  7. 闪烁计数器(WinSpectral,Wallac,PerkinElmer)
  8. 根据每个国家的法律,实验室准备了用于安全接收,储存,处理和处理放射性物质的所有必要设备。
  9. 盖革计数器(Ludlum Measurements,型号:3测量仪)

程序

注意:为了确定在何种条件下脂质合成随时间线性,制备不同的管并在37℃进行不同时间的反应。如果您正在评估改变给定蛋白质的脂质合成的能力,您还必须确定脂质合成与感兴趣的蛋白质的浓度成线性关系。
根据规则和管辖法律,进行实验的人员必须接受严格的放射性工作培训。

  1. 将放射性32 P掺入细胞/组织匀浆中
    1. 用所需的培养基在10cm培养皿上生长所需的细胞系,直到达到90%汇合。
      注意:在这一点上,如果你需要你的细胞培养 特殊情况或经过特殊治疗,您应该 满足您的需求的协议。
    2. 收获细胞与300 μlmilliQ水或使用匀浆器或ULTRA-TURRAX ?,如果样品 从组织获得,以获得蛋白质匀浆 到实验要求
    3. 在冰上超声,并使用Bradford方法或类似方法测量总蛋白浓度。
    4. 始终在冰上保持样品。
    5. 准备反应管。每个都必须包含最终卷 ?用1×反应缓冲液,3μCi的[γ-32 P] ATP和蛋白质 浓度为1.25mg/ml。考虑必须测量样品 至少一式三份。为此,首先在Kahn管中等分 达到所需蛋白质所需的蛋白质匀浆的量 浓度。然后,加入足够体积的反应缓冲液和 ?[γsup 32 P] ATP以达到上述浓度。正常 ?有几个条件需要测量,并考虑到 他们必须至少执行一式三份,我们找到它 方便准备所有管的主混合物,以避免移液 错误。为此,首先将蛋白质匀浆在等分试样中 不同的管,然后准备具有足够量的主混合物 反应缓冲液和[γ-32 P] ATP并将其等分在管中。最后, 用milliQ水达到最终体积。
      注意:为了简化,它 将有助于正常化所有样品的蛋白质浓度 待添加的主混合物的体积对于所有管是相同的。 此外,准备比主混音更大的音量 理论要求,补偿小体积损失或移液管 校准。使用milliQ水调整最终体积。
    6. 涡旋10秒。
    7. 在37℃下进行反应60分钟。
      注意:这时你可以通过冻结或你可以停止反应 继续纯化步骤。如果你停止反应 冷冻,样品必须放在沙坑中避免照射, 根据放射性物质的工作条件。
    8. 添加80 ?μlTCA-PTA 10-1(%w/v)以沉淀脂质和蛋白质。在这 点,你应该开始看到即将形成的白色 沉淀。涡旋10秒,并以3500rpm离心10分钟。

  2. 脂质沉淀和纯化
    1. 弃去上清液,其含有没有的游离的[γ 32 P] ATP ?并且涡旋,直到沉淀物被解聚。
    2. 加入1ml TCA-PTA 5-0.5(%w/v)并再次涡旋10秒。 离心并重复沉淀洗涤四次。丢弃 每轮后的上清液 注意:如果沉淀的量是 太小,你可以加入5微升脑匀浆,这将作为一个 ?载体(见图2)。


      图2. 预期金额的示例 ?沉淀(右管)后加入载体脑匀浆。 ?左边的管显示出沉淀物太小。

    3. 用milliQ水进行最终洗涤,以有利于脂质在有机溶剂中溶解(步骤B12)。
    4. 离心后,弃去最后一次洗涤的水上清液并涡旋,直到沉淀物解聚
    5. 加入1.5ml氯仿:甲醇2:1,涡旋10秒。的 氯仿溶解脂质,甲醇维持蛋白质 沉淀
    6. 在3,500rpm离心10分钟,以允许蛋白质沉淀和从可溶性脂质中分离。
    7. 转移1.3毫升含有标记的脂质的上清液 ?并在90℃水浴中干燥3小时或在室温下干燥 过夜。当转移上清液时,要小心,不要拉 ?沉淀的蛋白质,其可以用 P标记。
      警告:As 该步骤包括转移含氯仿的溶液, 应使用预先用氯仿平衡过的玻璃吸管 ?避免样品损失。
      注意:转移时损失0.2 ml 避免上清液干扰沉淀。

  3. 测量纯化脂质中的 P含量
    1. 加入1ml非水闪烁鸡尾酒,轻敲并涡旋10秒。
    2. 使用a确定脂质中掺入的32 P的水平 液闪烁计数器。反应必须在中进行 一式三份,最小。

  4. 数据分析
    1. 结果可以表示为绝对值(如图3所示) 或作为相对于作为100%的控制条件的相对值 纳入。

代表数据

图3是指示预期结果的类型的数据的代表性示例。在这种情况下,实验条件测量在c-Fos蛋白,其磷酸化形式或其突变体存在下的磷脂合成量。在这方面,结果表示为磷脂标记的量(Ferrero等人,2012)。


图3.通过c-Src磷酸化的c-Fos不激活磷脂合成。通过纯化的c-Src(Pc-Fos)磷酸化重组c-Fos,c-Fos的磷脂合成的活性,如先前所述(Gil等人,2004)检查c-Fos的磷酸模拟Y10/30E突变体和c-Fos Y10/30F的不可磷酸化突变体。在37℃下孵育60分钟。如先前所述进行P-磷脂定量(Guido和Caputto,1990)。结果表示为c.p.m。的掺入磷脂/mg蛋白质的平均值±s.d。的三个实验重复三次; *通过单向ANOVA分析确定的相对于对照(缓冲液)的P <0.002。 Y10/30F不与未加任何底物孵育的c-Src和c-Src一起作为对照。注意,在测定中c-Src的存在不改变磷脂合成。

笔记

如果在进行步骤A6中的反应之后决定冷冻,则不添加TCA-PTA 10-1(%w/v)溶液,因为匀浆可能聚集并与未掺入的[γ P] ATP分子,这可能导致错误的测量。

食谱

  1. 缓冲液HEPES(2M,pH7.5) 将476.6g HEPES溶解在800ml H 2 O中 用适当体积的浓NaOH将pH调节至7.5 将H sub 2 O添加到1L的最终体积中。
  2. 反应缓冲液(20x,20ml)

    试剂
    数量
    集中
    NaCl
    3.2726 g
    2,800 mM
    KCl
    0.1491克
    100 mM
    MgCl 2
    0.019克
    10 mM
    葡萄糖
    0.4036克
    112 mM
    HEPES缓冲液(2M,pH7.5) 12.8 ml

    加入milliQ水至最终体积为20ml ml 如果储存在-20℃,缓冲液可稳定6个月。
  3. TCA-PTA 10-1(%w/v)
    重量10克TCA和1克PTA,并加入milliQ水至终体积为100ml
  4. TCA-PTA 5-0.5(%w/v)
    等分50ml TCA-PTA 10-1,加milliQ水至终体积为100ml。
  5. 载体脑匀浆
    安乐死成年Wistar老鼠和消除大脑
    称重并制备1g组织/ml milliQ水的匀浆

致谢

我们感谢Beatriz L. Caputto博士有益的讨论,以适应和修改她以前的工作的协议(Guido和Caputto,1990)。我们也感谢CONICET和FONCYT的资金。

参考文献

  1. Guido,M.E。和Caputto,B.L。(1990)。 在暴露于光照或黑暗的鸡中对视网膜和视神经磷脂的标记。 J Neurochem 55(6):1855-1860。
  2. Ferrero,G.O.,Renner,M.L.,Gil,G.A.,Rodriguez-Berdini,L.and Caputto,B.L。(2014)。 c-Fos激活的核磷脂酰肌醇4,5-二磷酸酯[PtdIns(4,5) P(2)]促进全球转录变化。 Biochem J 461(3):521-530。
  3. Ferrero,G.O.,Velazquez,F.N.和Caputto,B.L。(2012)。 激酶c-Src和磷酸酶TC45协调调节c-Fos酪氨酸磷酸化和c-Fos磷脂合成激活能力。 Oncogene 31(28):3381-3391。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Rodriguez-Berdini, L. and Ferrero, G. O. (2016). A Technique for the Measurement of in vitro Phospholipid Synthesis via Radioactive Labeling. Bio-protocol 6(2): e1705. DOI: 10.21769/BioProtoc.1705.
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