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Measurement of Pyruvate in Drosophila Larvae Adipose Tissue with Commercial Kit   

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摘要:丙酮酸 (pyruvate) 是糖酵解进入三羧酸循环,以及糖异生的中心代谢产物,丙酮酸的水平与一些代谢类疾病相关。本文介绍了一种使用商业化的试剂盒检测果蝇幼虫脂肪组织丙酮酸的方法。

关键词: 脂肪体, 丙酮酸, 能量代谢

材料与试剂

  1. 移液枪头
  2. 96孔板
  3. 10 KDa蛋白filter (Amicon Ultra centrifugal filters 10K)
  4. 果蝇3龄幼虫
  5. 液氮
  6. 丙酮酸检测试剂盒 (Abcam, catalog number: ab65342)
  7. Brandford Reagent (Sigma, catalog number: B6916-500ML)
  8. BSA (阿拉丁, catalog number: A104912-100g)

仪器设备

  1. 移液枪
  2. 研磨器 (Koktes)
  3. -80 °C冰箱
  4. 酶标仪
  5. 离心机 (Eppendorf, model: 5415R)
  6. 摇床 (KANGJIAN, model: DSR-22)

实验步骤

  1. 样品准备,一组三个重复,每个重复需要fat body至少50只,解剖时放在冰上,将样品置于200 μl左右的pyruvate assay buffer中。样品最好使用新鲜组织,若不能及时完成可以用液氮速冻,置于-80 °C保存。
  2. 将样品在冰上研磨。取10 μl用于测蛋白,其余用于测丙酮酸。
  3. 用于测丙酮酸的样品先4 °C,12,000 × g离心2 min,取上清到10 KDa的filter去蛋白,4 °C,最大转速离心至少10 min (10 min可以得到不能全部过滤,但可以得到大于100 μl的样品)。
  4. 同时准备丙酮酸标准样。495 μl pyruvate assay buffer加5 μl丙酮酸标准样稀释到1 nmol/μl,再用1 nmol/μl丙酮酸标准样按下表梯度稀释:0,4,8,12,16,20 μl标准样分别加到100,96,92,88,84,80 μl pyruvate assay buffer,每孔加50 μl标准样,使得每孔0,2,4,6,8,10 nmol丙酮酸。
  5. 配制反应液。根据反应数,配制足够的Mix (反应液为样品数 + 标准样,Blank为样品数),每个反应液50 μl:
    反应液:46 μl pyruvate assay buffer + 2 μl pyruvate probe + 2 μl enzyme mix
    Blank: 48 μl pyruvate assay buffer + 2 μl pyruvate probe
  6. 将50 μl标准样96孔板,然后加入反应液。将过滤后的样品量 (大于100 μl,不够需提前稀释) 加2× 50 μl到96孔板中,分别加入50 μl反应液与Blank。
  7. 尽量避免气泡,在摇床混匀,室温反应30 min。检测OD570nm值。
  8. 同时准备测蛋白。先用pyruvate assay buffer,配制好BSA标准样。先配制浓度大的母液,再稀释2 mg/ml,1 mg/ml,0.5 mg/ml,0.25 mg/ml,0.125 mg/ml, 0 mg/ml,取10 μl反应。
  9. 将用于测蛋白的样品用pyruvate assay buffer稀释5倍,再4 °C,12,000 × g,离心2 min,取10 μl上清加入到200 μl Brandford反应液中,37 °C反应10 min。
  10. 取180 μl蛋白反应液上清加入96孔板,不要有气泡。用酶标仪检测OD595nm值。
  11. 根据丙酮酸和蛋白质的标准样制作标准曲线,并计算丙酮酸与蛋白量。
  12. 本试剂盒也可以根据荧光强度检测丙酮酸,且灵敏度更强,可用更少样品量检测。
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Copyright: © 2019 The Authors; exclusive licensee Bio-protocol LLC.
引用格式:许梦瑶, 黄勋. (2019). 果蝇幼虫脂肪组织丙酮酸 (Pyruvate) 检测 (试剂盒). Bio-101: e1010284. DOI: 10.21769/BioProtoc.1010284.
How to cite: Xu, M. Y. and Huang, X. (2019). Measurement of Pyruvate in Drosophila Larvae Adipose Tissue with Commercial Kit. Bio-101: e1010284. DOI: 10.21769/BioProtoc.1010284.
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