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Detection of Wnt5 in Media Conditioned by Mouse Embryonic Fibroblast
检测小鼠胚胎成纤维细胞调节培养基中的Wnt5   

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Abstract

This protocol describes the procedure of visualizing secreted Wnt5 protein in serum free media via western blotting. This procedure can also be used to visualize other secreted proteins larger than 10,000 daltons. The work presented in this paper visualizes Wnt5 secreted by mouse embryonic fibroblast (MEF), but can be adapted to other cell lines including those transiently transfected by plasmids.

Keywords: Wnt5(wnt5), Detection(检测), Conditioned Media(空调媒体)

Materials and Reagents

  1. 10 cm Petri dishes
  2. 50 ml Beckel centrifuge tubes
  3. 1.5 ml microcentrifuge tubes
  4. Amicon Ultra-15 centrifugal filter
  5. 0.45 µm immobilon-FL PVDF (EMD Millipore, catalog number: IPFL00010 )
  6. Wild type MEF and Rab8a-/- MEF cell lines
  7. MEF isolation and phenotypic analysis was previously described (Das et al., 2015)
  8. DEME with glucose, L-glutamine and sodium pyruvate (Mediatech, catalog number: 10-013 )
  9. Fetal bovine serum (FBS) (Sigma-Aldrich, catalog number: F2442-500ML )
  10. Pen-strep (Thermo Fisher Scientific, GibcoTM, catalog number: 15140-122 )
  11. Lactalbumin hydrolysate solution (50x) (Sigma-Aldrich, catalog number: 58901C-100ML )
  12. NaN3
  13. NaF
  14. Na3VO4
  15. PMSF
  16. DTT
  17. Nonidet® P40 substitute (Sigma-Aldrich, catalog number: 74385-1L )
  18. Bio-Rad protein assay dye reagent concentrate (Bio-Rad Laboratories, catalog number: 500-0006 )
  19. Tween® 20 (Thermo Fisher Scientific, Fisher Scientific, catalog number: BP337-500 )
  20. Sodium chloride (NaCl)
  21. Potassium chloride (KCl)
  22. Disodium hydrogen phosphate (Na2HPO4)
  23. Potassium dihydrogen phosphate (KH2PO4)
  24. Protease inhibitor cocktail tablets (Sigma Aldrich, catalog number: 11873580001 )
  25. Skim milk powder (EMD Millipore, catalog number: 1.15363.0500 )
  26. Amersham ECL Western blotting detection reagents (GE Healthcare, catalog number: RPN2209 )
  27. Amersham ECL Rabbit IgG, HRP-linked whole Ab (from donkey) (GE Healthcare, catalog number: NA934V-1ML )
  28. Wnt5a/b (C27E8) Rabbit mAB (Cell Signaling Technology, catalog number: 2530S )
  29. Histone H3 (D1H2) XP® Rabbit mAb (Cell Signaling Technology, catalog number: 4499S )
  30. 10% FBS DMEM (see Recipes)
  31. 1x LAH DMEM (see Recipes)
  32. Non-denaturing lysis buffer (see Recipes)
  33. 1% Tween 20 in PBS (PBST) (see Recipes)
  34. 5% skim milk in PBST (see Recipes)
  35. 1x running buffer (see Recipes)
  36. 1x transfer buffer (see Recipes)

Equipment

  1. Thermo IEC Centra CL3R Bench-model, Refrigerated centrifuge
  2. Avanti J-26 XP centrifuge (Beckman Coulter, model: Avanti J-26XP )
  3. Beckman JA-25.50 rotor (Beckman Coulter, model: JA-25.50 )
  4. Bio-Rad Mini-PROTEAN Tetra Cell (Bio-Rad Laboratories, model: 1658005EDU )
  5. XCell SureLock® Mini-Cell and XCell II blot module (Thermo Fisher Scientific, NovexTM, catalog number: EI0002 )
  6. QSonica XL-2000 (Qsonica, model: XL-2000 )
  7. Ultrospec 2100® UV-Visible spectrophotometer (Biochrom, model: 80-2112-21 )

Procedure

  1. Cell culture/transfection
    1. Culture wild type MEF and Rab8a-/- MEF cell lines in DMEM + 10% FBS until 90-95% confluent in a 10 cm Petri dish.
    2. Replace DMEM + 10% FBS with DMEM supplemented with 12 ml of 1x lactalbumin hydrolysate solution (LAH).
    3. Monitor the morphology of cells over the next 48 h.
    4. After 48 h, collect the conditioned media in a 50 ml centrifuge tube and spin down at 10,000 x g at 4 °C for 10 min. Transfer the supernatant into a clean tube and immediately proceed with step B1 for maximum yield. Supernatant can be frozen and used after thawing but is generally not recommended. 
    5. Wash Petri dish with 1 ml of 1x PBS. Repeat wash.
    6. Subsequently add 1 ml of 1x lysis buffer to Petri dish and lay flat on ice for 15 min.
    7. Keeping the Petri dish on ice, collect the cell lysate using a cell scraper and transfer to a 1.5 ml microcentrifuge tube.
    8. Sonicate cell lysate briefly (no more than 1 or 2 sec at a time for 3 cycles) and then centrifuge at 13,300 x g at 4 °C for 10 min.
    9. Transfer supernatant to a clean microcentrifuge tube and store at -80 °C.

  2. Centrifugation of conditioned media
    1. Take the conditioned media and add it to the Amicon Ultra-15 centrifugal filter.
    2. Centrifuge the filtration device at 4,000 x g at room temperature for 90 min.
    3. Use a 200 µl pipette to recover the concentrate. For maximum recovery, collect the concentrate immediately. Expect roughly, 180 µl of concentrated media from collection.
    4. Save the concentrate at -80 °C. Aliquot can remain frozen for up to a year.

  3. Western blot detection
    1. Measure protein concentrate using Bradford assay (Bio-Rad reagent protocol). Use lysis buffer as a blank for protein measurements.
    2. Prepare 30 µg of total protein samples in 1x LDS sample buffer.
    3. Run samples in 10% SDS-PAGE gel at 80 V and immediately stop when dye runs off gel.
    4. Transfer protein onto a 0.45 µm PVDF membrane. Setup a transfer apparatus and run at 300 mA for 90 min.
    5. After transferring, take the PVDF membrane and block using 5% skim milk in PBST. Leave the membrane for blocking at room temperature on a shaker for 1 h.
    6. Subsequently, take the membrane and incubate overnight in a 1:1,000 dilution of anti-mWnt5a/b antibody at 4 °C. Dilute antibody in 5% skim milk in PBST.
    7. After 16 h of incubation in primary antibody, wash membrane in PBST and place on a shaker for 10 min. Repeat two more times for a total of three washes.
    8. Incubate with anti-rabbit antibody at a 1:2,000 dilution at room temperature for 1 h. Dilute antibody in 5% skim milk in PBST.
    9. Wash membrane in PBST and place on a shaker for 10 min. Repeat two more times for a total of three washes.
    10. Apply ECL reagent to membrane for 5 min.
    11. Proceed to expose x-ray film for 30 sec and develop.

Representative data



Figure 1. Detection of Wnt5a in conditioned media. Cell lysates and conditioned media from Rab8a+/+ and Rab8a-/- MEF visualized using anti-mWnt5a primary antibody. Histone3 was used as a control to determine contamination from cell lysate in media samples.

Recipes

  1. 10% FBS DMEM
    950 ml DMEM with 4.5 g/L glucose, L-glutamine & sodium pyruvate
    50 ml fetal bovine serum
    5 ml pen-strep
  2. 1x LAH DMEM (50 ml)
    49 ml DMEM with 4.5 g/L glucose, L-glutamine & sodium pyruvate
    1 ml lactalbumin hydrolysate solution (50x)
  3. Non-denaturing lysis buffer
    50 mM Tris-HCl (pH 7.5)
    150 mM NaCl
    10 mM EDTA
    0.02% NaN3
    50 mM NaF
    1 mM Na3VO4
    0.5% Np40 substitute
    1 mM PMSF
    0.5 mM DTT
    Protease inhibitor cocktail tablets (1tablet for 10mL)
  4. 0.1% Tween 20 in PBS (PBST) (1 L)
    1 ml Tween® 20
    8 g NaCl
    0.2 g KCl
    1.44 g Na2HPO4
    0.24 g KH2PO4
    Dissolved in 1,000 ml MilliQ water, and pH adjusted to 7.4 using NaOH.
  5. 5% skim milk in PBST
    5 g skim milk powder dissolved in 100 ml PBST.
  6. 1x running buffer
    3.0275 g Tris base
    14.4 g glycine
    10 g sodium dodecyl sulfate
    Dissolved in up to 1,000 ml of MilliQ water.
  7. 1x transfer buffer
    14.4 g glycine
    3.02 g Tris base
    Dissolved in 700 ml of MilliQ water
    Add 200 ml methanol once completely dissolved.

Acknowledgments

This work was supported by National Institutes of Health (NIH) grants [DK102934, DK085194, DK093809, CA178599]. NG is also supported by a Research Scholar Grant [RSG-15-060-01-TBE] from the American Cancer Society.

References

  1. Das, S., Yu, S., Sakamori, R., Vedula, P., Feng, Q., Flores, J., Hoffman, A., Fu, J., Stypulkowski, E., Rodriguez, A., Dobrowolski, R., Harada, A., Hsu, W., Bonder, E. M., Verzi, M. P. and Gao, N. (2015). Rab8a vesicles regulate Wnt ligand delivery and Paneth cell maturation at the intestinal stem cell niche. Development 142(12): 2147-2162.

简介

该协议描述了通过免疫印迹观察无血清培养基中分泌的Wnt5蛋白的过程。 该程序还可用于显现大于10,000道尔顿的其他分泌的蛋白质。 本文提出的工作可视化由小鼠胚胎成纤维细胞(MEF)分泌的Wnt5,但可以适应于其他细胞系,包括由质粒瞬时转染的那些。

关键字:wnt5, 检测, 空调媒体

材料和试剂

  1. 10厘米培养皿
  2. 50 ml Beckel离心管
  3. 1.5 ml微量离心管
  4. Amicon Ultra-15离心过滤器
  5. 0.45μmimmobilon-FL PVDF(EMD Millipore,目录号:IPFL00010)
  6. 野生型MEF和Rab8a -/- MEF细胞系
  7. 之前描述了MEF分离和表型分析(Das等人,2015)
  8. DEME与葡萄糖,L-谷氨酰胺和丙酮酸钠(Mediatech,目录号:10-013)
  9. 胎牛血清(FBS)(Sigma-Aldrich,目录号:F2442-500ML)
  10. Pen-strep(Thermo Fisher Scientific,Gibco TM ,目录号:15140-122)
  11. 乳白蛋白水解产物溶液(50x)(Sigma-Aldrich,目录号:58901C-100ML)
  12. NaN 3
  13. NaF
  14. Na 3 VO 4
  15. PMSF
  16. DTT
  17. Nonidet P40替代品(Sigma-Aldrich,目录号:74385-1L)
  18. Bio-Rad蛋白测定染料试剂浓缩物(Bio-Rad Laboratories,目录号:500-0006)
  19. Tween 20(Thermo Fisher Scientific,Fisher Scientific,目录号:BP337-500)
  20. 氯化钠(NaCl)
  21. 氯化钾(KCl)
  22. 磷酸氢二钠(Na 2 HPO 4)
  23. 磷酸二氢钾(KH 2 PO 4)
  24. 蛋白酶抑制剂混合物片剂(Sigma Aldrich,目录号:11873580001)
  25. 脱脂乳粉(EMD Millipore,目录号:1.15363.0500)
  26. Amersham ECL Western印迹检测试剂(GE Healthcare,目录号:RPN2209)
  27. Amersham ECL兔IgG,HRP连接的整个Ab(来自驴)(GE Healthcare,目录号:NA934V-1ML)
  28. Wnt5a/b(C27E8)兔mAB(Cell Signaling Technology,目录号:2530S)
  29. 组蛋白H3(D1H2)XP兔mAb(Cell Signaling Technology,目录号:4499S)
  30. 10%FBS DMEM(参见配方)
  31. 1x LAH DMEM(参见配方)
  32. 非变性裂解缓冲液(参见配方)
  33. 1%Tween 20的PBS(PBST)(见Recipes)
  34. PBST中的5%脱脂牛奶(参见配方)
  35. 1x运行缓冲区(参见配方)
  36. 1x传输缓冲区(请参阅配方)

设备

  1. Thermo IEC Centra CL3R台式,冷冻离心机
  2. Avanti J-26 XP离心机(Beckman Coulter,型号:Avanti J-26XP)
  3. Beckman JA-25.50转子(Beckman Coulter,型号:JA-25.50)
  4. Bio-Rad Mini-PROTEAN Tetra Cell(Bio-Rad Laboratories,型号:1658005EDU)
  5. XCell SureLock Mini-Cell和XCell II印迹模块(Thermo Fisher Scientific,Novex TM ,目录号:EI0002)
  6. QSonica XL-2000(Qsonica,型号:XL-2000)
  7. Ultrospec 2100紫外 - 可见分光光度计(Biochrom,型号:80-2112-21)

程序

  1. 细胞培养/转染
    1. 在DMEM + 10%FBS中培养野生型MEF和Rab8a -/- MEF细胞系,直到在10cm陪替氏培养皿中90-95%汇合。
    2. 用补充有12ml1X乳清蛋白水解产物溶液(LAH)的DMEM替换DMEM + 10%FBS。
    3. 监测细胞的形态在接下来的48小时。
    4. 48小时后,将条件培养基收集在50ml离心管中并在4℃下以10,000×g离心10分钟。将上清液转移到干净的管中,并立即进行步骤B1最大产量。上清液可以在解冻后冷冻和使用,但通常不推荐使用。
    5. 洗涤培养皿1毫升1×PBS。重复清洗。
    6. 随后向培养皿中加入1ml 1x裂解缓冲液,在冰上平放15分钟
    7. 保持培养皿在冰上,使用细胞刮刀收集细胞裂解液,并转移到1.5毫升微量离心管。
    8. 超声处理细胞裂解液(每次不超过1或2秒,进行3个循环),然后在13,300×g,4℃离心10分钟。
    9. 将上清液转移到干净的微量离心管中,在-80℃下保存
  2. 条件培养基离心
    1. 取条件培养基并将其加入Amicon Ultra-15离心过滤器
    2. 在室温下将过滤装置以4,000×g离心90分钟
    3. 使用200μl移液器回收浓缩液。为了最大限度地恢复,立即收集浓缩物。大约收集180μl浓缩的培养基。
    4. 将浓缩液保存在-80°C。等分试样可以保存冷冻最多一年。

  3. Western印迹检测
    1. 使用Bradford测定(Bio-Rad试剂方案)测量蛋白质浓缩物。使用裂解缓冲液作为空白用于蛋白质测量
    2. 在1×LDS样品缓冲液中制备30μg总蛋白样品
    3. 在80V下在10%SDS-PAGE凝胶中运行样品,并且当染料脱离凝胶时立即停止
    4. 将蛋白转移到0.45μmPVDF膜上。设置转印设备,并在300 mA下运行90分钟。
    5. 转移后,取PVDF膜并用PBST中的5%脱脂乳封闭。使膜在室温下在振荡器上阻断1小时
    6. 随后,取膜并在4℃下在1:1000稀释的抗-mWnt5a/b抗体中孵育过夜。在PBST中的5%脱脂乳中稀释抗体
    7. 在第一抗体孵育16小时后,在PBST中洗涤膜,并置于振荡器上10分钟。重复两次,共三次洗涤。
    8. 与抗兔抗体在室温下1:2,000稀释孵育1小时。在PBST中的5%脱脂乳中稀释抗体
    9. 在PBST中洗涤膜,并置于振荡器上10分钟。重复两次,共三次洗涤。
    10. 应用ECL试剂到膜5分钟。
    11. 继续曝光X光胶片30秒并显影。

代表数据



图1.在条件培养基中检测Wnt5a。来自Rab8a +/+和Rab8a -/- MEF的细胞裂解物和条件培养基用抗 - -mWnt5a一抗。组蛋白3用作对照以确定培养基样品中细胞裂解物的污染。

食谱

  1. 10%FBS DMEM
    950ml具有4.5g/L葡萄糖,L-谷氨酰胺&丙酮酸钠 50ml胎牛血清
    5ml pen-strep
  2. 1x LAH DMEM(50ml)
    49ml具有4.5g/L葡萄糖的DMEM,L-谷氨酰胺&丙酮酸钠 1ml乳清蛋白水解产物溶液(50x)
  3. 非变性裂解缓冲液
    50mM Tris-HCl(pH7.5) 150mM NaCl 10 mM EDTA
    0.02%NaN 3
    50mM NaF 1mM Na 3 VO 4 sub。
    0.5%Np40替代品
    1mM PMSF
    0.5 mM DTT
    蛋白酶抑制剂鸡尾酒片(1片,10mL)
  4. 0.1%Tween 20的PBS(PBST)(1L)中 1ml Tween ? 20
    8克NaCl
    0.2克KCl
    1.44g Na 2 HPO 4
    0.24g KH 2 PO 4 sub/
  5. PBST中的5%脱脂牛奶
    将5g脱脂奶粉溶于100ml PBST中
  6. 1x运行缓冲区
    3.0275克Tris碱
    14.4g甘氨酸
    10g十二烷基硫酸钠 溶解在最多1000毫升MilliQ水中。
  7. 1x传输缓冲区
    14.4g甘氨酸
    3.02克Tris碱
    溶于700ml MilliQ水中
    加入200ml甲醇,一旦完全溶解

致谢

这项工作由国家卫生研究院(NIH)授予[DK102934,DK085194,DK093809,CA178599]支持。 NG也得到美国癌症协会的研究学者资助[RSG-15-060-01-TBE]的支持。

参考文献

  1. Das,S.,Yu,S.,Sakamori,R.,Vedula,P.,Feng,Q.,Flores,J.,Hoffman,A.,Fu,J.,Stypulkowski,E.,Rodriguez, Dobrowolski,R.,Harada,A.,Hsu,W.,Bonder,EM,Verzi,MP and Gao,N.(2015)。  Rab8a囊泡调节Wnt配体递送和Paneth细胞在肠干细胞巢的成熟发育 142 12):2147-2162。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Flores, J. and Gao, N. (2016). Detection of Wnt5 in Media Conditioned by Mouse Embryonic Fibroblast. Bio-protocol 6(20): e1971. DOI: 10.21769/BioProtoc.1971.
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