搜索

Protein Extraction from Drosophila Embryos and Ovaries
从果蝇胚胎和卵巢提取蛋白质   

引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

参见作者原研究论文

本实验方案简略版
eLIFE
Apr 2014

Abstract

Here we provide the description of protocols to efficiently obtain protein extracts from embryos and ovaries of Drosophila melanogaster. These protocols are routinely applied in our laboratory and are based on two techniques: either embryos or ovaries are homogenized using a pestle and then the soluble proteins separated by centrifugation, or embryos are individually lysed by needle manipulation. The latter technique allows the use of small embryo numbers and the selection of specific developmental stages (Guilgur et al., 2014).

Keywords: Drosophila (果蝇), Protein extracts (蛋白提取物), Embryos (胚胎), Ovaires (ovaires)

Materials and Reagents

  1. Phosphate buffered saline tablets (Sigma-Aldrich, catalog number: P4417-100TAB )
  2. Commercial bleach solution
  3. Tween 20 (Sigma-Aldrich, catalog number: P5927 )
  4. Tris-base (DBH Prolabo, catalog number: 33621.26 0)
  5. NaCl (Panreac Applichem, catalog number: 121659.1211 )
  6. EDTA (Sigma-Aldrich, catalog number: E6758 )
  7. DL-dithiothreitol (DTT) (Sigma-Aldrich, catalog number: 43819 )
  8. NP-40 (IGEPAL CA-630) (Sigma-Aldrich, catalog number: I8896 )
  9. Sodium fluoride (NaF) (Fluka, catalog number: 71519 )
  10. NaOH (sodium hydroxide pellets) (Panreac, catalog number: 131687 )
  11. Agar-agar (Nzytech, Agar-agar, catalog number: MB14801 )
  12. Sugar (commercial)
  13. Apple juice (commercial)
  14. Niapagine (Dutscher, Niapagine, catalog number: 789063 )
  15. Complete EDTA-free protease inhibitor tablets (Roche Diagnostics, catalog number: 04693159001 )
  16. Sample buffer (2x Laemmli sample buffer) (Sigma-Aldrich, catalog number: S3401 )
  17. Commercial fresh baker´s yeast paste
  18. NB lysis buffer (see Recipes)
  19. 1 M Tris-HCl (see Recipes)
  20. 500 mM EDTA (see Recipes)
  21. 10% NP-40 (see Recipes)
  22. 0.5 M NaF (see Recipes)
  23. 1 M DTT (see Recipes)
  24. Apple juice plates (see Recipes)

Equipment

  1. Containers (dark tip boxes to increase the contrast with the white embryos)
  2. Cell strainer (70 μm nylon cell strainer) (BD Biosciences, Falcon®, catalog number: 352350 )
  3. 1.5 ml tubes
  4. 1.5 μl pestles (Kimble Chase, catalog number: 749521-1590 )
  5. Paint brush number 4
  6. Needles (0.8 x 25 mm) (Terumo, catalog number: NN-2125R )
  7. Tweezers (Fine Science Tools, Dumont #5 )
  8. Refrigerated centrifuge (Eppendorf, model: 5424R )
  9. Fly cages  
  10. Small Petri dishes (Sarstedt, catalog number: 83.1801.002 )
  11. Filters (Acrodisc Syringe Filters 0.2 μm Supor Membrane) (Pall, catalog number: PN 4612 )

Procedure

  1. For protein extraction from ovaries
    1. Ovary dissection
      1. Rear female flies alongside a small fraction (1:3) of males in food supplemented with fresh baker yeast paste for one to two days prior to dissection. This will stimulate oogenesis and lead to bigger ovaries (with increased numbers of late developmental stages).
      2. Inactivate anaesthetized flies by decapitation.
      3. Dissection technique (under stereoscope and using dissection plate and a pair of tweezers): For each fly, place the organism in a drop of 1x PBS and hold it in place by applying gentle pressure at the level of the upper thorax. Using the tweezers in the free hand tug gently at the lower part of the abdomen (ovipositor region) until the cuticle starts to detach from the fly, exposing the internal organs. Isolate ovaries from adjoining tissues and organs and transfer them to ice cold 1x PBS while dissecting the remaining flies (avoid keeping the ovaries in the 1x PBS solution for periods longer than 30 min) (see Video 1 for a visual description).

        Video 1. Drosophila ovary dissection

    2. Ovary protein extraction by sample homogenization
      1. Transfer the isolated ovaries to a 1.5 ml tube containing 200 μl of ice-cold NB lysis buffer.
      2. Manually homogenize samples using a pestle. Homogenization should ensure the complete breakdown of the tissue. If pestles are to be reused, wash them thoroughly with distilled water before processing other samples.
      3. Centrifuge for 20 sec at ~10,000 rcf (4 °C) to settle down at the bottom of the tube the unprocessed tissue.
      4. Repeat manual homogenization of the centrifuged material.
      5. Centrifuge for 3 min at ~20,000 rcf (4 °C).
      6. Transfer the supernatant to a new 1.5 ml tube, avoiding the upper lipid layer.
      7. Repeat this centrifugation process (steps A2 e-f) two more times.
      8. Quantify protein concentration and dilute to the final concentration (dependent on the requirements of downstream applications).
      9. Dilute final concentration with an equal volume of 2x Laemmli sample buffer.
      10. Heat samples for 5 min at 100 °C and immediately freeze them at -20 °C after a quick centrifuge spin-down. Extracts can be stored at -20 °C until necessary.

  2. For protein extraction from embryos
    1. Embryo collection and processing
      1. For one to two days prior to embryo collection rear male and female flies on collection cages with standard apple juice agar plates supplemented with fresh baker yeast paste (Figure 1 A).
      2. Start the collection by placing a clean apple juice agar plate on the cage. Let flies lay eggs for a given time interval.
      3. While egg laying is taking place prepare the 5 individual containers for the subsequent processing of the embryos. Containers with the following solutions are required: 0.1% Tween 20 (in water), 50% commercial bleach (in water) and deionized water (3 containers). Place a collection basket (cell strainer) into the container with the 0.1% Tween 20 solution (starting point) (Figure 1A).
      4. Collect embryos from the agar plate using a small paintbrush and place them in the partially immersed basket.
      5. Gently stir the collection basket to wash the embryos. Dry the base of the collection basket in a paper tissue before transferring it to the subsequent container.
      6. Transfer the embryos in the collection basket to the container with the 50% commercial bleach solution. Incubate for 5 min with gentle, periodic stirring. The purpose of this step (dechorionation) is to remove the chorionic membranes which constitute the eggshell covering the embryos. The dechorionated embryos will become hydrophobic and will float on the surface of the bleach solution (Figure 1B).
      7. Transfer the embryos in the collection basket to a new container with deionized water. Wash for 2 min and repeat twice using each time new containers. Before starting the washes and also when transferring between the water containers dry the base of the collection basket in a paper tissue.
        Note: Embryos can be stored at this step by transferring them to a 1.5 ml tube (remove excess water) that will be flash frozen in liquid nitrogen prior to storage at -80 °C.


        Figure 1. Set up for embryonic protein extraction. Fly collection cages with standard apple juice agar plates and solution containers set up (A). Dechorionated embryos float on the surface of the bleach solution (B). Manually select embryos collected in a 0.5 ml tube lid (C1). Punctured embryo total extract (C2). Total extract collection by mixing with Laemmli sample buffer (C3). Embryo collection before pestle homogenization (Arrowhead 1, D1). Embryonic soluble protein extract after homogenization and centrifugation (Arrowhead 2, D2).

    2. Embryo total protein extracts

      Manual selection and needle homogenization protocol
      1. After dechorionation, manually select embryos with sharp tweezers or a fine paintbrush and transfer them to a previously sectioned 0.5 ml tube lid (for a minimum of 10 embryos per lid) (Figure 1C1).
      2. Dry as much as possible the embryos by absorbing the excess water with a dry paintbrush.
      3. Individually puncture each embryo using a needle (Figure 1C2).
      4. Gently mix the resulting lysate with 10 µl of 1x Laemmli sample buffer (Figure 1C3). Transfer the solution to a 1.5 ml collection tube.
        Note: The volume of sample Laemmli buffer is according to the number of embryos used (1 µl per embryo).
      5. Heat samples for 5 min at 100 °C and immediately freeze them at -20 °C after a quick centrifuge spin-down. Extracts can be stored at -20 °C until necessary.

      Pestle homogenization protocol
      1. After dechorionation, transfer embryos (20 μl volume) to a 1.5 ml tube containing 200 μl of ice-cold NB lysis buffer (Figure 1D1).
        Note: This will correspond approximately to between 1 to 2 μg/μl of final protein concentration. Manually homogenize embryos using a pestle (~10 strokes). Homogenization should ensure the complete breakdown of the tissue. If pestles are to be reused, wash them thoroughly before processing other samples.
      2. Centrifuge for 20 sec at ~10,000 rcf (4 °C) to settle down at the bottom of the tube the unprocessed tissue.
      3. Repeat manual homogenization of the centrifuged material.
      4. Centrifuge for 3 min at ~20,000 rcf (4 °C).
      5. Transfer the supernatant to a new 1.5 ml tube, avoiding the upper lipid layer (Figure 1D2).
      6. Repeat this centrifugation process (steps B2 i-j) two more times.
      7. Quantify protein concentration and dilute to the final concentration (dependent on the requirements of downstream applications).
      8. Dilute final concentration with an equal volume of 2x Laemmli sample buffer.
      9. Heat samples for 5 min at 100 °C and immediately freeze them at -20 °C after a quick centrifuge spin-down. Extracts can be stored at -20 °C until necessary.

Recipes

  1. NB buffer
    Initial concentration
    Volume
    Final concentration
    1 M NaCl
    7.5 ml
    150 mM NaCl
    1 M Tris-HCl (pH 7.5)
    2.5 ml
    50 mM Tris-HCl (pH 7.5)
    500 mM EDTA (pH 8.0)
    200 µl
    2 mM EDTA
    10% NP-40
    500 µl
    0.1% NP-40
    Add ddH2O to final volume of 50 ml
    Sterilized by filtration (0.2 µm filter)
    Make 10 ml Aliquots and store at -20 °C
    Before use add to the 10 ml aliquot: 10 µl of 1 M DTT, 200 µl of 0.5 M NaF and dissolve one Complete EDTA-free tablet to the solution
  2. 1 M Tris-HCl (pH 7.5)
    Dissolve 157.6 g of Tris-HCl to ~800 ml of ddH2O
    Adjust pH to 7.5 with NaOH
    Add ddH2O to final volume of 1,000 ml
    Sterilized by filtration (0.2 µm filter)
    Stored at RT
  3. 500 mM EDTA (pH 8.0)
    Weigh 73.06 g of EDTA to ~400 ml of ddH2O
    Adjust pH slowly to 8.0 with NaOH - EDTA dissolves when pH approaches 8
    Add ddH2O to final volume of 500 ml
    Sterilized by filtration (0.2 µm filter)
    Stored at RT
  4. 10% NP-40
    Dilute 10 ml of NP-40 to ddH2O in a final volume of 100 ml
    Sterilized by filtration (0.2 µm filter)
    Stored at RT
  5. 0.5 M NaF
    Dissolve 2.0995 g of NaF to ddH2O in a final volume of 100 ml
    Sterilized by Filtration (0.2 µm filter)
    Aliquot and stored at -20 °C
  6. 1 M DTT
    Dissolve 1.5425 g of DTT to ddH2O in a final volume of 10 ml in the fume hood
    Sterilized by filtration (0.2 µm filter)
    Aliquot and stored at -20 °C
  7. Apple juice plates (1 L)
    Weigh Agar-agar in a big plastic beaker 19.5 g
    Add to the Agar-agar ddH2O 500 ml
    Mix everything very well
    Place the beaker in microwave until boiling
    Wait for the medium to cool to 50 °C, stirring from time to time to avoid the formation of a film on the surface
    Weigh sugar in an aluminum foil 20 g
    Then add to the dissolve Agar-agar: The sugar, Apple juice 250 ml, Niapagin 10% 5 ml and ddH2O 250 ml
    1 L of apple juice medium → 100 small plates
    Store the plates at 4 °C no more than 30 days

Acknowledgments

We like to thank Paulo Navarro-Costa for critical reading of manuscript and Rui Martinho for his supervi-sion. Funding: FCT-Fundaçao para a Ciencia e Tecnologia (Portugal): Leonardo Gastón Guilgur, SFRH/BPD/47957/2008. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

References

  1. Guilgur, L. G., Prudencio, P., Sobral, D., Liszekova, D., Rosa, A. and Martinho, R. G. (2014). Re-quirement for highly efficient pre-mRNA splicing during Drosophila early embryonic development. Elife 3: e02181.

简介

FLP / FRT系统是基于将重组酶(翻转酶-FLP)靶向指定为翻转酶识别靶标(FRT)位点的特定DNA区域的定点重组技术。最初在酿酒酵母中鉴定,酵母FLP酶及其FRT重组靶点成功转移到果蝇中的每个主要染色体臂(Golic和Lindquist,1989)。这提供了以受控方式在发育过程中在体内介导有丝分裂重组的能力[在Theodosiou和Xu(1998)中修订]。有丝分裂重组事件的受控诱导通常通过在热休克(hs)启动子的控制下表达FLP来进行。这允许在特定发育时间窗口表达高FLP水平。携带这些遗传标记的FLP / FRT染色体的菌株大大增强了我们在种系和体细胞果蝇组织中研究基因功能的能力。在这里我们描述两种不同的方案:一种用于诱导和鉴定卵巢中的纯合突变体克隆,另一种用于产生雌性种系突变体,用于分析母体对胚胎发生的影响。

关键字:果蝇, 蛋白提取物, 胚胎, ovaires

材料和试剂

  1. 磷酸盐缓冲盐水片(Sigma-Aldrich,目录号:P4417-100TAB)
  2. 商业漂白溶液
  3. 吐温20(Sigma-Aldrich,目录号:P5927)
  4. Tris-base(DBH Prolabo,目录号:33621.260)
  5. NaCl(Panreac Applichem,目录号:121659.1211)
  6. EDTA(Sigma-Aldrich,目录号:E6758)
  7. DL-二硫苏糖醇(DTT)(Sigma-Aldrich,目录号:43819)
  8. NP-40(IGEPAL CA-630)(Sigma-Aldrich,目录号:I8896)
  9. 氟化钠(NaF)(Fluka,目录号:71519)
  10. NaOH(氢氧化钠颗粒)(Panreac,目录号:131687)
  11. 琼脂(Nzytech,Agar-agar,目录号:MB14801)
  12. 糖(商业)
  13. 苹果汁(商业)
  14. Niapagine(Dutscher,Niapagine,目录号:789063)
  15. 完整的无EDTA蛋白酶抑制剂片剂(Roche Diagnostics,目录号:04693159001)
  16. 样品缓冲液(2x Laemmli样品缓冲液)(Sigma-Aldrich,目录号:S3401)
  17. 商业新鲜面包酵母膏
  18. NB裂解缓冲液(参见配方)
  19. 1 M Tris-HCl(参见配方)
  20. 500 mM EDTA(见配方)
  21. 10%NP-40(见配方)
  22. 0.5 M NaF(参见配方)
  23. 1 M DTT(参见配方)
  24. 苹果汁盘(见配方)

设备

  1. 容器(暗盒提高与白色胚胎的对比度)
  2. 细胞过滤器(70μm尼龙细胞滤器)(BD Biosciences,Falcon ,目录号:352350)
  3. 1.5 ml管
  4. 1.5μl杵(Kimble Chase,目录号:749521-1590)
  5. 画笔号4
  6. 针(0.8×25mm)(Terumo,目录号:NN-2125R)
  7. 镊子(Fine Science Tools,Dumont#5)
  8. 冷冻离心机(Eppendorf,型号:5424R)
  9. 飞笼
  10. 小培养皿(Sarstedt,目录号:83.1801.002)
  11. 过滤器(Acrodisc注射器过滤器0.2μmSupor膜)(Pall,目录号:PN 4612)

程序

  1. 用于从卵巢中提取蛋白质
    1. 卵巢解剖
      1. 后女性沿着一小部分飞行                              (1:3)的男性在食品中补充新鲜的面包酵母膏一个                              到解剖前两天。 这将刺激卵子发生和铅                              到更大的卵巢(随着晚期发育的数量增加                              阶段)。
      2. 通过断头灭活麻醉的苍蝇。
      3. 解剖技术(在立体镜下和使用解剖板和a                             对镊子):对于每次飞,放置在一滴1x PBS的生物                             并通过在水平处施加轻柔的压力将其保持在适当位置                             上胸部。使用镊子在自由手中轻轻地拖动                             下腹部(产卵区),直到角质层开始                             从飞行中分离,暴露内脏。分离卵巢                             从邻接的组织和器官,并将其转移到冰冷的1×PBS                             同时解剖剩余的苍蝇(避免保持卵巢在                             1x PBS溶液,时间长于30分钟)(见视频1 for a                             视觉描述)。

        视频1.果蝇卵巢解剖
        <! - [if!IE]> - <! - <![endif] - >

        要播放视频,您需要安装较新版本的Adobe Flash Player。

        获取Adobe Flash Player

        <! - [if!IE]> - >
        <! - <![endif] - >

    2. 通过样品匀浆的卵巢蛋白提取
      1. 将隔离的卵巢转移到含有200μl冰冷的NB裂解缓冲液的1.5ml管中
      2. 使用杵手动匀浆样品。 均质化应该                              确保组织的完全破坏。 如果要杵                              重复使用,在加工前用蒸馏水彻底清洗                              其他样品。
      3. 在约10,000 rcf(4℃)下离心20秒,以在管的底部沉降未处理的组织。
      4. 重复手动匀浆离心的材料。
      5. 在〜20,000rcf(4℃)下离心3分钟。
      6. 转移上清液到一个新的1.5毫升管,避免上脂质层。
      7. 重复该离心过程(步骤A2e-f)两次以上
      8. 定量蛋白质浓度并稀释至最终浓度                              (取决于下游应用的要求)。
      9. 用等体积的2x Laemmli样品缓冲液稀释终浓度
      10. 在100℃下加热样品5分钟,立即在-20℃下冷冻                              °C后快速离心机下降。 提取物可储存于-20°C                              直到需要。

  2. 用于从胚胎中提取蛋白质
    1. 胚胎收集和处理
      1. 前一到两天                              胚胎收集后面男性和女性飞行在收集笼子与                              标准苹果汁琼脂平板上补充新鲜面包酵母                              (图1A)。
      2. 开始收集通过将一个干净的苹果汁琼脂板在笼子上。 让苍蝇在给定的时间间隔内产卵。
      3. 在进行产卵时,准备5个单独的容器                              用于胚胎的后续处理。 容器与                              需要以下溶液:0.1%吐温20(在水中),50% 商业漂白剂(在水中)和去离子水(3个容器)。放置a                             收集篮(细胞过滤器)装入带有0.1%                             吐温20溶液(起始点)(图1A)
      4. 使用小画笔从琼脂板收集胚胎,并将其放置在部分浸没的篮子。
      5. 轻轻搅拌收集篮以洗涤胚胎。干燥基座                             的收集篮在纸组织中,然后转移到它                             后续容器。
      6. 转移胚胎在集合中                             篮子装入50%商业漂白溶液的容器中。                             孵育5分钟,温和,定期搅拌。这个的目的                             步骤(去氧化)是去除绒毛膜                             构成覆盖胚胎的蛋壳。 dechorionated胚胎                             将变得疏水并且将漂浮在漂白剂的表面上 溶液(图1B)。
      7. 转移胚胎在集合中                             篮子用去离子水装入新容器。洗涤2分钟和                             重复两次使用每次新容器。开始洗涤前                             并且当在水容器之间转移时干燥的基部                             收集篮在纸巾中。
        注意:胚胎可以                                 在此步骤中通过将其转移至1.5ml管中(除去过量)                                 水),其在储存之前将在液氮中快速冷冻                                 -80℃。


        图1.设置胚胎蛋白提取                             收集笼与标准苹果汁琼脂板和溶液                             集装箱设置(A)。脱钙胚胎浮在表面                             漂白溶液(B)。手动选择收集在0.5毫升管中的胚胎                             盖(C1)。穿刺胚提取物(C2)。总提取物收集 通过与Laemmli样品缓冲液(C3)混合。 胚胎收集之前                              杵均质化(箭头1,D1)。 胚胎可溶性蛋白                              提取物均质化和离心(箭头2,D2)。

    2. 胚总蛋白提取物

      手动选择和针同质化协议
      1. 脱壳后,用锋利的镊子手动选择胚胎                              一个精细的画笔,并将它们转移到先前切片0.5毫升                              管盖(每个盖至少10个胚胎)(图1C1)。
      2. 通过用干的油漆刷吸收过量的水尽可能多地干燥胚胎。
      3. 使用针单独刺穿每个胚胎(图1C2)。
      4. 轻轻混合所得裂解物与10μl的1x Laemmli样品                              缓冲液(图1C3)。 将溶液转移到1.5ml收集管。
        注意:Laemmli缓冲液样品的体积根据所使用的胚胎数量(每个胚胎1μl)。
      5. 在100℃下加热样品5分钟,立即在-20℃下冷冻                              °C后快速离心机下降。 提取物可储存于-20°C                              直到需要。

      杵同质化方案
      1. 后                             脱壳,将胚胎(20μl体积)转移到1.5ml管中                             含有200μl冰冷的NB裂解缓冲液(图1D1)。
        注意:                                 这将大致对应于最终的1至2μg/μl                                 蛋白质浓度。使用杵手工匀浆胚胎(〜10                                 笔画)。均质化应确保完全破碎                                 组织。如果要重复使用杵,请先将其彻底清洗                                 处理其他样品。
      2. 在约10,000 rcf(4℃)下离心20秒,以在管的底部沉降未处理的组织。
      3. 重复手动匀浆离心的材料。
      4. 在〜20,000rcf(4℃)离心3分钟。
      5. 转移上清液到一个新的1.5毫升管,避免上脂质层(图1D2)。
      6. 重复该离心过程(步骤B2 i-j)两次以上
      7. 定量蛋白质浓度并稀释至最终浓度                              (取决于下游应用的要求)。
      8. 用等体积的2x Laemmli样品缓冲液稀释终浓度
      9. 在100℃下加热样品5分钟,立即在-20℃下冷冻                              °C后快速离心机下降。 提取物可储存于-20°C                              直到需要。

食谱

  1. NB缓冲器
    初始浓度

    最终浓度
    1 M NaCl
    7.5 ml
    150mM NaCl
    1 M Tris-HCl(pH 7.5)
    2.5 ml
    50mM Tris-HCl(pH7.5)
    500mM EDTA(pH8.0) 200μl
    2mM EDTA
    10%NP-40
    500微升
    0.1%NP-40
    将ddH 2 O加到最终体积为50ml的
    中 过滤灭菌(0.2μm过滤器)
    制成10毫升等分试样并储存在-20°C
    使用前加入10 ml的等分试样:10μl的1M DTT,200μl的0.5 M NaF,并溶解一个完整的无EDTA片剂到溶液中
  2. 1 M Tris-HCl(pH 7.5)
    将157.6g Tris-HCl溶解于〜800ml ddH 2 O中
    用NaOH调节pH至7.5 将ddH <2> O加入到最终体积为1000ml
    过滤灭菌(0.2μm过滤器)
    存储在RT
  3. 500mM EDTA(pH8.0) 称量73.06g EDTA至〜400ml ddH 2 O 2 / 用NaOH缓慢调节pH至8.0 - 当pH接近8时,EDTA溶解。
    将ddH 2 O加至最终体积为500ml
    过滤灭菌(0.2μm过滤器)
    存储在RT
  4. 10%NP-40
    将10ml NP-40稀释至ddH 2 O,最终体积为100ml
    过滤灭菌(0.2μm过滤器)
    存储在RT
  5. 0.5 M NaF
    在最终体积100ml中将2.0995g NaF溶解到ddH 2 O中。
    过滤灭菌(0.2μm过滤器)
    等分并储存在-20°C
  6. 1 M DTT
    在通风橱中将1.5425g DTT溶解于ddH 2 O中,最终体积为10ml。
    过滤灭菌(0.2μm过滤器)
    等分并储存在-20°C
  7. 苹果汁盘(1升)
    称量琼脂在一个大塑料烧杯中19.5克
    加到琼脂 - 琼脂ddH 2 O 500ml
    混合一切非常好
    将烧杯置于微波直到沸腾
    等待介质冷却至50°C,不时搅拌,以避免在表面上形成薄膜
    在铝箔20克
    中称重糖 然后加入到溶解的琼脂中:糖,苹果汁250ml,Niapagin 10%5ml和ddH 2 O 250ml
    1升苹果汁介质→100小板
    将平板在4℃保存不超过30天

致谢

我们要感谢保罗纳瓦罗 - 科斯塔对手稿的批判性阅读,以及Rui Martinho为他的监督。 资金:FCT-Fundaçaopara a Ciencia e Tecnologia(葡萄牙):LeonardoGastónGuilgur,SFRH/BPD/47957/2008。 资助者在研究设计,数据收集和解释或提交出版工作的决定方面没有任何作用。

参考文献

  1. Guilgur,L.G.,Prudencio,P.,Sobral,D.,Liszekova,D.,Rosa,A.and Martinho,R.G。(2014)。 在果蝇早期胚胎期间需要高效的前mRNA剪接的需求 。 3:e02181。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright Prudêncio and Guilgur. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Prudêncio, P. and Guilgur, L. G. (2015). Protein Extraction from Drosophila Embryos and Ovaries. Bio-protocol 5(9): e1459. DOI: 10.21769/BioProtoc.1459.
  2. Guilgur, L. G., Prudencio, P., Sobral, D., Liszekova, D., Rosa, A. and Martinho, R. G. (2014). Re-quirement for highly efficient pre-mRNA splicing during Drosophila early embryonic development. Elife 3: e02181.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。

当遇到任何问题时,强烈推荐您通过上传图片的形式提交相关数据。