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p53, is known as the guardian of the genome and as such requires exquisite regulation not only of its abundance but also its activity. The abundance of p53 can be modulated at the level of transcription, translation, and also via its degradation.

This protocol involves 35S metabolic labelling of newly synthesized proteins followed by a period of chase with "cold" media. Samples are harvested and p53 immunopreciptiated, separated by SDS PAGE and the levels of 35S labelled p53 determined. By comparing the level of 35S p53 at 0 h to those "chased" with cold media (e.g. 60 min) provides an indication of the rate of p53 turnover.

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Analyze p53 degradation by 35S p53 Pulse Chase Analysis
采用35S p53 脉冲追踪分析 p53降解

生物化学 > 蛋白质 > 免疫检测
作者: Megan Astle
Megan AstleAffiliation: Growth Control and Differentiation Program, Peter MacCallum Cancer Centre, Melbourne, Australia
Bio-protocol author page: a142
Richard Pearson
Richard PearsonAffiliation: Growth Control and Differentiation Program, Peter MacCallum Cancer Centre, Melbourne, Australia
Bio-protocol author page: a143
 and Katherine Hannan
Katherine HannanAffiliation: Growth Control and Differentiation Program, Peter MacCallum Cancer Centre, Melbourne, Australia
For correspondence: kate.hannan@petermac.org
Bio-protocol author page: a144
Vol 2, Iss 21, 11/5/2012, 4784 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.283

[Abstract] p53, is known as the guardian of the genome and as such requires exquisite regulation not only of its abundance but also its activity. The abundance of p53 can be modulated at the level of transcription, translation, and also via its degradation.

This protocol involves 35S metabolic labelling of newly synthesized proteins followed by a period of chase with "cold" media. Samples are harvested and p53 immunopreciptiated, separated by SDS PAGE and the levels of 35S labelled p53 determined. By comparing the level of 35S p53 at 0 h to those "chased" with cold media (e.g. 60 min) provides an indication of the rate of p53 turnover.

[Abstract] 该实验方案的中文版正在准备中...

Materials and Reagents

  1. Dulbecco’s Modified Eagle Medium (DMEM) methionine and cysteine free media (1x) (Life Technologies, Invitrogen™, catalog number: 21013-024 )
  2. Dialysed fetal bovine serum (FBS) (Life Technologies, Invitrogen™, catalog number: 26400-036 )
  3. Express 35S protein labelling mix (PerkinElmer, catalog number: NEG 0720008MC )
  4. Cell lifter (Corning Incorporated, catalog number: 3008 )
  5. Protein A sepharose (Zymed, catalog number: 10-1042 )
  6. p53 DO-1 (Santa Cruz, catalog number: SC-126 )
  7. DC Protein assay kit (Bio-Rad Laboratories, catalog number: 500-0116 )
  8. Bio-Rad safe stain solution (Bio-Rad Laboratories, catalog number: 161-0786 )
  9. L-glutatmine or Glutamax (Life Technologies, Invitrogen™, catalog number: 35050-061 )
  10. cOmplete Ultra tablets (Roche Applied Science, catalog number: 05 892 988 001
  11. PhosSTOP tablets (Roche Applied Science, catalog number: 049068450001 )
  12. NP-40 (IGEPAL, catalog number: CA-630 )
  13. Beta mercapotethanol
  14. Bromophenol blue
  15. Complete media (see Recipes)
  16. Starvation media (see Recipes) 
  17. Dulbecco’s PBS (see Recipes)
  18. 1x lysis buffer (see Recipes)
  19. 2x SLB buffer (see Recipes)
  20. Washed protein A sepharose (see Recipes)

Equipment

  1. Bioruptor sonicator (Diagenode)
  2. Macsmix tube rotator (Miltenyi Biotech GmbH) or standard Rotating wheel
  3. Heat block
  4. SDS–polyacrylamide gel electrophoresis (PAGE) running equipment
  5. Orbital shaker
  6. 3 mm whatman paper and plastic wrap
  7. Gel Dryer and pump
  8. Storage phosphor screen (Molecular Dynamics) and Typhoon Trio (GE Healthcare)

Software

  1. Image Quant software (Molecular Dynamics)

Procedure

  1. Labelling
    1. Cells were plated in complete media at 350,000 per 100 mm plate 48 h before harvesting (this will be cell type dependent, aim for 70% confluence at day of harvest).
    2. Aspirate media and wash the cells gently with room temperature (RT) PBS (10 ml per 100 mm plate).
    3. Remove PBS and replace with warmed (37 °C) starvation media (6 ml per 100 mm plate).
    4. Return cells to the incubator for 1 h.
    5. Add 200 μCi of Express 35S protein labelling mix (18 μl per dish) directly into the media and swirl.
    6. Return to the incubator for 30 min.
    7. Either:
      1. Harvest (0 h)
      2. OR remove the media and wash the plate twice with 5 ml of warmed complete media.
      3. Add complete media and return plates to the incubator for the designated period of time (15 min, 30 min, 45 min, 60 min) before harvest.

  2. Harvest (Keep samples cold at all times)
    1. Place plates on ice and remove the media.
    2. Wash plates gently twice with 5 ml of ice cold PBS, then drain the plate on its side and remove any residual PBS.
    3. Add 500 μl of lysis buffer with 0.5% NP-40 and scrapped with a cell lifter.
    4. Pippette lysate into a 1.5 ml Eppendorf tube.
    5. Sonicate lysate for 5 min with a cycle of 30 sec on then 30 sec off at 4 °C using the Bioruptor.
    6. Spin sample at 11,000 x g for 10 min at 4 °C.
    7. Transfer supernatant to a new tube keeping them on ice.
    8. Determine the protein concentration of sample using the DC Bio-Rad assay as per manufactures instructions with BSA as a control. Samples were assayed in triplicate.

  3. p53 Immunopreciptiation
    1. Equivalent amounts of protein were added to each tube and made up to a final volume of 400 μl with lysis buffer.
    2. To each sample add 40 μl of washed protein A sepharose to preclear the sample.
      Note: Beads are in a 50% slurry so the final bed volume is 20 μl; use a tip which has the last 20% cut off the end to generate a larger bore size, pipette up and down at least 5 times to insure the beads are not stuck in the tip.

    3. Rotate sample for 1 h at 4 °C on wheel.
    4. Spin sample at 800 x g for 2 min at 4 °C.
    5. Transfer supernatant to fresh tube and:
      1. Remove 20 μl to keep as "input" for loading control purposes.
      2. Add 40 μl of a 50% slurry of washed protein A sepharose with 0.3 μg of p53 or control antibody.
    6. Tumble overnight at 4 °C.
    7. Spin at 800 x g for 2 min at 4 °C.
    8. Remove supernatant and wash beads with 600 μl of lysis buffer with 0.1% NP-40 three times (flick tube to resuspend beads don’t pipette them).
    9. Spin sample at 800 x g for 2 min at 4 °C.
    10. Remove supernatant and wash beads with 1 ml of Lysis buffer with no NP-40 two more times.
    11. Spin sample at 800 x g for 2 min at 4 °C.
    12. Remove all supernatant and add 40 μl of 2.5x SLB buffer.
    13. Boil beads at 95 °C for 10 min in a heatblock.
    14. Cool on ice for 5 min.
    15. Boil for a further 5 min in a heat block.
    16. Pulse spin at max speed for 30 sec.
    17. Transfer supernatant to new tube (~50 μl/sample).
    18. Load input, immunoprecipitation and markers on a 10% PAG with 4% stacker, 1.5 mm thick, 10 well comb, then run the gel at 100 V for about 2 h (stop when dye is almost at the bottom of the gel).
    19. Remove gel from the plates and wash gel twice with ddH2O.
    20. Stain the gel in Bio-Rad safe stain solution for 1 h while gently rotating on an orbital shaker.
    21. Replace the stain and incubate for a further 20 min while gently rotating on an orbital shaker.
    22. Remove gel from stain and put in a fresh container then add water to destain, refresh the water 2-4 times over 1 h.
    23. Carefully place the gel on two layers of 3 mm paper, cover with glad wrap and dry using a Bio-Rad gel drier at 80 °C for ~2 h (till no longer sticky).
    24. Place gel by the phosphoimager screen overnight then analyze with the Typhoon Trio.
    25. Quantitate the bands using the Image Quant software (Molecular Dynamics).

Recipes

  1. Complete media
    1x DMEM
    10% FBS
    1x Glutamax
  2. Starvation media
    Methionine and Cysteine free DMEM
    15% dialysed FBS
    1x Glutamax
  3. Dulbecco’s PBS (for 1 L)
    0.2 g KCl
    0.2 g KH2PO4
    2.3 g Na2HPO4
    8 g NaCl
  4. 1x lysis buffer
    Use stock solutions to make the following final concentrations
    50 mM Tris (pH 8.0)
    150 mM NaCl
    5 mM EDTA
    1x complete (protease inhibitors)
    1x PhosphoSTOP
    Plus 0.5% or 0.1% NP-40 (v/v): Add fresh to the buffer using a 10% stock of NP-40, keep cool to help resuspend the NP-40 (IGEPAL CA-630)
  5. 2x SLB
    500 mM Tris (pH 6.8)
    20% SDS
    20% Beta mercapotethanol
    0.1% bromophenol blue
  6. Washed protein A sepharose
    The protein A sepharose is stored in ethanol which needs to be removed.
    1. Remove 2.5x the volume you need for the experiment
    2. Pulse spin at 4 °C (Let centrifuge get to maximum speed and then stop)
    3. Remove supernatant and add 5x the volume in 6 a) of lysis buffer plus 0.5% NP-40
    4. Repeat pulse spin and repeat wash
    5. Remove supernatant and add 0.5x the volume in 6a) of lysis buffer to generate a 50% slurry

Acknowledgments

This protocol was originally published in its brief version in Astle et al. (2012). This work was supported by grants from the National Health and Medical Research Council (NHMRC) of Australia to RDH (NHMRC Nos. 166908 and 251688) and to RBP (NHMRC Nos. 509087 and 400116) and from Cancer Council Victoria to RBP. RDH and RBP are NHMRC Senior Research Fellows. We acknowledge the Victorian Centre for Functional Genomics for the use of equipment and advice. We thank Professor Stephen Jane and Loretta Cerruti for assistance with retrovirus production.

References

  1. Astle, M. V., Hannan, K. M., Ng, P. Y., Lee, R. S., George, A. J., Hsu, A. K., Haupt, Y., Hannan, R. D. and Pearson, R. B. (2012). AKT induces senescence in human cells via mTORC1 and p53 in the absence of DNA damage: implications for targeting mTOR during malignancy. Oncogene 31(15): 1949-1962.

材料和试剂

  1. Dulbecco's Modified Eagle Medium(DMEM)甲硫氨酸和不含半胱氨酸的培养基(1x)(Life Technologies,Invitrogen TM,目录号:21013-024)
  2. 透析胎牛血清(FBS)(Life Technologies,Invitrogen TM,目录号:26400-036)
  3. Express S蛋白标记混合物(PerkinElmer,目录号:NEG 0720008MC)
  4. 电池升降器(Corning Incorporated,目录号:3008)
  5. 蛋白A琼脂糖(Zymed,目录号:10-1042)
  6. p53 DO-1(Santa Cruz,目录号:SC-126)
  7. DC蛋白测定试剂盒(Bio-Rad Laboratories,目录号:500-0116)
  8. Bio-Rad安全染色溶液(Bio-Rad Laboratories,目录号:161-0786)
  9. L-谷氨酰胺或Glutamax(Life Technologies,Invitrogen TM,目录号:35050-061)
  10. cOmplete Ultra片剂(Roche Applied Science,目录号:05 892 988 001)
  11. PhosSTOP片剂(Roche Applied Science,目录号:049068450001)
  12. NP-40(IGEPAL,目录号:CA-630)
  13. β巯基乙醇
  14. 溴酚蓝
  15. 完成媒体(见配方)
  16. 饥饿媒体(请参阅食谱)
  17. Dulbecco的PBS(参见食谱)
  18. 1x裂解缓冲液(见配方)
  19. 2x SLB缓冲液(见配方)
  20. 洗涤的蛋白A琼脂糖(见配方)

设备

  1. Bioruptor超声波仪(Diagenode)
  2. Macsmix管旋转器(Miltenyi Biotech GmbH)或标准旋转轮
  3. 热块
  4. SDS-聚丙烯酰胺凝胶电泳(PAGE)运行设备
  5. 轨道振动器
  6. 3毫米whatman纸和塑料包装
  7. 凝胶干燥器和泵
  8. 储存荧光屏(Molecular Dynamics)和Typhoon Trio(GE Healthcare)

软件

  1. 图像定量软件(Molecular Dynamics)

程序

  1. 标签
    1. 在收获前48小时,将细胞以350,000/100mm平板接种在完全培养基中(这将是细胞类型依赖性的,目标是在收获日70%汇合)。
    2. 吸出培养基,并用室温(RT)PBS(10ml/100mm板)轻轻洗涤细胞
    3. 取出PBS,并更换为温热(37°C)饥饿培养基(每100 mm平板6 ml)
    4. 将细胞返回培养箱1小时。
    5. 将200μCi的Express 35 S蛋白标记混合物(每个培养皿18μl)直接加入培养基并旋转。
    6. 返回孵育器30分钟。
    7. 或者:
      1. 收获(0小时)
      2. 或取出介质,用5毫升温热的完全培养基洗涤板两次
      3. 在收获前,将指定的时间(15分钟,30分钟,45分钟,60分钟)添加完整培养基并将培养板放回培养箱。

  2. 收获(保持样品始终冷)
    1. 将板放在冰上,取出介质。
    2. 用5ml冰冷的PBS轻轻洗涤平板两次,然后将平板排在其侧面,并除去任何残留的PBS
    3. 加入500μl含0.5%NP-40的裂解缓冲液,并用细胞提取器报废
    4. 将pippette裂解物倒入1.5ml Eppendorf管中
    5. 超声处理裂解液5分钟,周期为30秒,然后使用Bioruptor在4℃下30秒关闭。
    6. 在4℃下,以11,000×g /分钟旋转样品10分钟
    7. 将上清转移到新管中,使其保持在冰上
    8. 使用DC Bio-Rad测定根据制造商说明书使用BSA作为对照确定样品的蛋白质浓度。 样品一式三份进行测定

  3. p53免疫沉淀
    1. 将等量的蛋白质加入每个管中,并用裂解缓冲液补足至400μl的最终体积
    2. 向每个样品中加入40μl洗涤的蛋白A琼脂糖凝胶以预清洗样品。
      注意:珠子在50%浆液中,因此最终床体积为20μl; 使用具有最后20%截止端的尖端产生较大的孔径,向上和向下移动吸头至少5次,以确保珠子不会粘在尖端中。
    3. 在4℃下在轮上旋转样品1小时。
    4. 在4℃下在800×g下旋转样品2分钟
    5. 转移上清液到新鲜管和:
      1. 取出20μl,保持为"输入"加载控制目的
      2. 加入40μl50%的洗涤的蛋白A琼脂糖凝胶与0.3μgp53或对照抗体的浆液
    6. 在4℃下翻滚过夜。
    7. 在4℃下以800×g离心2分钟。
    8. 取出上清液,用600μl含有0.1%NP-40的裂解缓冲液洗涤珠子三次(轻弹管重悬的珠子不吸取它们)。
    9. 在4℃下在800×g下旋转样品2分钟
    10. 除去上清液,用1ml没有NP-40的裂解缓冲液洗珠子两次以上
    11. 在4℃下在800×g下旋转样品2分钟
    12. 取出所有上清液,加入40μl2.5x SLB缓冲液
    13. 在95℃下在加热块中煮沸10分钟
    14. 在冰上冷却5分钟。
    15. 在加热块中再煮沸5分钟。
    16. 以最大速度脉冲旋转30秒。
    17. 将上清转移到新试管中(〜50μl/样品)
    18. 在具有4%堆叠器,1.5mm厚,10孔梳子的10%PAG上的负载输入,免疫沉淀和标记,然后在100V下运行凝胶约2小时(当染料几乎在凝胶底部时停止) br />
    19. 从板上除去凝胶并用ddH 2 O洗涤凝胶两次
    20. 将凝胶在Bio-Rad安全染色溶液中染色1小时,同时在轨道摇床上轻轻旋转
    21. 更换染色剂,并在轨道摇床上轻轻旋转的同时孵育20分钟
    22. 从染色中除去凝胶,并放入新鲜的容器中,然后加水脱色,在1小时内将水重新洗2-4次
    23. 小心地将凝胶放置在两层3mm的纸上,盖上快速包装,使用Bio-Rad凝胶干燥器在80℃下干燥〜2小时(直到不再粘稠)。
    24. 通过phosphoimager屏幕放置凝胶过夜,然后用台风三重奏分析
    25. 使用Image Quant软件(Molecular Dynamics)对带进行定量。

食谱

  1. 填写媒体
    1x DMEM
    10%FBS
    1x Glutamax
  2. 饥饿媒体
    甲硫氨酸和半胱氨酸游离DMEM
    15%透析FBS
    1x Glutamax
  3. Dulbecco's PBS(1L)
    0.2克KCl
    0.2g KH 2 PO 4 sub/
    2.3g Na 2 HPO 4
    8克NaCl
  4. 1x裂解缓冲液
    使用储备溶液制备以下最终浓度
    50mM Tris(pH8.0) 150mM NaCl 5 mM EDTA
    1x完全(蛋白酶抑制剂)
    1x PhosphoSTOP
    加入0.5%或0.1%NP-40(v/v):使用NP-40的10%储备液向缓冲液中加入新鲜,保持冷却以帮助重悬NP-40(IGEPAL CA-630)
  5. 2x SLB
    500mM Tris(pH6.8)
    20%SDS
    20%β-巯基乙醇 0.1%溴酚蓝
  6. 洗涤的蛋白A琼脂糖
    将蛋白A琼脂糖储存在需要除去的乙醇中。
    1. 删除实验所需的2.5倍体积
    2. 脉冲旋转在4°C(让离心机达到最大速度,然后停止)
    3. 除去上清液,加入6倍体积的裂解缓冲液加0.5%NP-40的体积为5倍
    4. 重复脉冲旋转并重复洗涤
    5. 除去上清液,加入在6a)裂解缓冲液中的0.5倍体积以产生50%浆料

致谢

该协议最初以Astle等人的简要版本发布。(2012)。这项工作得到了澳大利亚国家卫生和医学研究委员会(NHMRC)向RDH(NHMRC Nos.166908和251688)和RBP(NHMRC Nos.509087和400116)以及从维多利亚癌症委员会到RBP的资助。 RDH和RBP是NHMRC高级研究员。我们承认维多利亚功能基因组学中心使用设备和建议。我们感谢Stephen Jane和Loretta Cerruti教授协助进行逆转录病毒生产。

参考文献

  1. Astle,M.V.,Hannan,K.M.,Ng,P.Y.,Lee,R.S.,George,A.J.,Hsu,A.K.,Haupt,Y.,Hannan,R.D.and Pearson,R.B。(2012)。 AKT在缺乏DNA损伤的情况下通过mTORC1和p53诱导人类细胞衰老:对mTOR靶向的影响在恶性肿瘤期间。癌基因 31(15):1949-1962。
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How to cite this protocol: Astle, M., Pearson, R. and Hannan, K. (2012). Analyze p53 degradation by 35S p53 Pulse Chase Analysis. Bio-protocol 2(21): e283. DOI: 10.21769/BioProtoc.283; Full Text



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