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Protocol for Preparation of Nuclear Protein from Mouse Lungs
小鼠肺部细胞核蛋白制备法   

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参见作者原研究论文

本实验方案简略版
Cancer Cell
Apr 2013

Abstract

This protocol describes how to extract nuclear protein from mouse lungs, including tissue preparation, stepwise lysis of cells and centrifugal isolation of nuclear protein fraction. This is an efficient method to get comparable nuclear protein extracts from lung tissues.

Materials and Reagents

  1. Mice
  2. DNase I (100 mg/ml) (Sigma-Aldrich, catalog number: DN25 )
  3. 1x PBS
  4. HEPES (Life Technologies, Gibco®, catalog number: 15630-080 )
  5. Magnesium chloride solution (Sigma-Aldrich, catalog number: M1028 )
  6. Potassium chloride solution (Sigma-Aldrich, catalog number: 60121 )
  7. Nonidet P40 (NP-40) (Roche Diagnostics, catalog number: 11332473001 )
  8. Fetal calf serum (FCS)
  9. Bovine serum albumin (BSA)
  10. 0.25% trypsin (Life Technologies, Gibco®, catalog number: 15050 )
  11. CD45 MicroBeads kit (Miltenyi Biotec, catalog number: 130-052-301 )
  12. Cytoplasmic extract buffer (see Recipes)
  13. Nuclear extract buffer (see Recipes)

Equipment

  1. Tweezers and scissors
  2. 26 G1/2 needles (BD, catalog number: 309659 )
  3. Cell strainer (100 μm and 40 μm) (BD Biosciences, Falcon®, catalog numbers: 352360 and 352340 )
  4. 10 ml and 1 ml syringe (BD, catalog numbers: 309604 and 305111 )
  5. Filter tips (Eppendorf)
  6. 15 ml Corning tubes (Corning)
  7. DNase, RNase-free Eppendorf tubes (Eppendorf)
  8. Tissue culture dishes (60 x 15 mm style) (BD Biosciences, Falcon®, catalog number: 353002 )
  9. MACS device (Miltenyi Biotec, model: 0 16210 )

Procedure

  1. For each group, 2-3 mice at the age of 10-12 weeks old (about 25-30 g per mouse) were sacrificed and the pulmonary vasculatures were perfused with PBS until the lungs turned white.
    1. After the mice have been euthanized with CO2 exposure, open the chest widely, taking care not to puncture the lungs.
    2. In brief, the sacrificed mouse is placed in the supine position and secured to the table. Prepare a 10 ml 1x PBS in a syringe with a number 22 needle connected.
    3. After the right femoral artery is cut, introduce the needle into the left heart ventricles and slowly inject the PBS until the lungs are well perfused. Meanwhile the blood-PBS mixture flow out through the cut femoral artery.
  2. Lungs were collected, minced with scissors into small pieces (∼1 mm), and then incubated in 2 ml of 0.25% trypsin with DNase I (100 mg/ml) at 37 °C for 30-45 min with continuous mixing. FCS at 1% final concentration was added to inactivate trypsin and was chilled on ice.
    Note: In all subsequent steps lung preparations and solutions were kept at 4 °C.
  3. Lung homogenates were adjusted to 20 ml with PBS containing 0.05% BSA and DNase I (100 mg/ml) filtered through gauze followed by filtration through nylon filters with 100 μm and 40 μm, respectively. The cell concentration is kept at 1 x 106 to 1 x 107 per ml after filtration.
  4. The final filtrates were centrifuged at 300 x g for 10 min at 4 °C.
  5. Then, CD45+ cell depletion (remove hematopoietic cells) was performed following the manufacturer’s protocol (Miltenyi Biotec).
    1. Briefly listed the protocol as below. After resuspending cell pellet in 90 µl of buffer per 107 total cells, add 10 µl of CD45 micro beads. Then mix well and incubate for 15 min at 4-8 °C.
    2. Wash cells with 2 ml of and centrifuge at 300 x g for 10 min. Pepette off supernatant completely.
    3. Proceed to magnetic separation using Miltenyi magnetic device and collect unlabeled cells which pass through the column. This is the CD45- lung cells.
  6. Finally, the nuclear and cytoplasmic proteins from purified lung cells were to be prepared in the following steps.
  7. The lung cells were washed with cold PBS twice and were collected by spinning at 450 x g for 5 min.
  8. Then, 200-500 μl of cytoplasmic extract buffer plus protease inhibitor were added into the cell pellets.
  9. The pellets were gently mixed, kept on ice for 5 min, and then centrifuged at 3,000 x g for 5 min.
  10. The supernatants were collected as the cytoplasmic extract. Normally the final protein concentrations are around 2.5-4 µg/µl.
  11. The pellets were washed once with 500 μl of cytoplasmic extract buffer, suspended in 100-200 μl of nuclear extract buffer, gently mixed on ice for 5 min, and centrifuged at 14,000 x g for 5 min.
  12. The supernatants were collected as the nuclear extract. We usually monitor the purity of extracted proteins by running western blot with antibodies against HDAC-1 or histone 3 as nuclear control and Betta actin as cytoplasmic protein control.

Recipes

  1. Cytoplasmic extract buffer
    10 mM KCl
    10 mM Hepes (pH 7.9)
    3 mM MgCl2
    1.0% NP-40
  2. Nuclear extract buffer
    400 mM KCl
    10 mM Hepes (pH 7.9)
    3 mM MgCl2
    1.0% NP-40

Acknowledgments

We thank Yinling Hu, Robert H. Wiltrout and Jami Willette-Brown from Frederick National Laboratory for Cancer Research for their support and suggestion on the experimental design. This work was supported by the National Cancer Institute (ZIA BC 011212 and ZIA BC 011391 to Y.H.).

References

  1. Xiao, Z., Jiang, Q., Willette-Brown, J., Xi, S., Zhu, F., Burkett, S., Back, T., Song, N. Y., Datla, M., Sun, Z., Goldszmid, R., Lin, F., Cohoon, T., Pike, K., Wu, X., Schrump, D. S., Wong, K. K., Young, H. A., Trinchieri, G., Wiltrout, R. H. and Hu, Y. (2013). The pivotal role of IKKalpha in the development of spontaneous lung squamous cell carcinomas. Cancer Cell 23(4): 527-540.

简介

该协议描述如何从小鼠肺提取核蛋白,包括组织制备,逐步裂解细胞和核蛋白级分的离心分离。 这是从肺组织获得可比较的核蛋白提取物的有效方法。

材料和试剂

  1. 小鼠
  2. DNase I(100mg/ml)(Sigma-Aldrich,目录号:DN25)
  3. 1x PBS
  4. HEPES(Life Technologies,Gibco ,目录号:15630-080)
  5. 氯化镁溶液(Sigma-Aldrich,目录号:M1028)
  6. 氯化钾溶液(Sigma-Aldrich,目录号:60121)
  7. Nonidet P40(NP-40)(Roche Diagnostics,目录号:11332473001)
  8. 胎牛血清(FCS)
  9. 牛血清白蛋白(BSA)
  10. 0.25%胰蛋白酶(Life Technologies,Gibco ,目录号:15050)
  11. CD45 MicroBeads试剂盒(Miltenyi Biotec,目录号:130-052-301)
  12. 细胞质提取缓冲液(参见配方)
  13. 核提取缓冲液(见配方)

设备

  1. 镊子和剪刀
  2. 26 G1/2针(BD,目录号:309659)
  3. 细胞过滤器(100μm和40μm)(BD Biosciences,Falcon ,目录号:352360和352340)
  4. 10ml和1ml注射器(BD,目录号:309604和305111)
  5. 过滤提示(Eppendorf)
  6. 15ml康宁管(Corning)
  7. DNase,无RNA酶的Eppendorf管(Eppendorf)
  8. 组织培养皿(60×15mm样式)(BD Biosciences,Falcon ,目录号:353002)
  9. MACS装置(Miltenyi Biotec,型号:016210)

程序

  1. 对于每组,处死2-3只年龄为10-12周龄(小鼠约25-30g)的小鼠,并用PBS灌注肺血管直到肺变白。
    1. 在用CO 2 2暴露使小鼠安乐死后,广泛地打开胸部,注意不刺穿肺。
    2. 简言之,将牺牲的小鼠放置在仰卧位并且 固定到桌子上。 准备10毫升1×PBS在一个注射器与数字 22针连接。
    3. 右股动脉切开后,将针插入 左心室,并缓慢注射PBS,直到肺部良好   灌注。 同时,血液-PBS混合物通过切口流出 股动脉
  2. 收集肺,用剪刀剪成小片(〜1mm),然后在2ml的0.25%胰蛋白酶中与DNA酶I(100mg/ml)在37℃下温育30-45分钟,连续混合。加入1%终浓度的FCS以灭活胰蛋白酶并在冰上冷却 注意:在所有后续步骤中,将肺制剂和溶液保持在4℃。
  3. 将肺匀浆用含有0.05%BSA和DNase I(100mg/ml)的PBS调节至20ml,通过纱布过滤,然后分别通过100μm和40μm的尼龙过滤器过滤。过滤后细胞浓度保持在1×10 6至1×10 7个/ml。
  4. 将最终滤液在4℃下以300xg离心10分钟。
  5. 然后,按照制造商的方案(Miltenyi Biotec)进行CD45 +细胞清除(去除造血细胞)。
    1. 简要列出如下协议。 在将细胞沉淀重悬于90μl缓冲液/10μL总细胞后,加入10μlCD45微珠。 然后混匀,在4-8℃下孵育15分钟
    2. 用2ml的洗涤细胞并在300×g离心10分钟。 完全除去上清液。
    3. 使用Miltenyi磁性装置进行磁分离 收集通过柱的未标记细胞。 这是CD45-   肺细胞。
  6. 最后,来自纯化的肺细胞的核和细胞质蛋白质将在以下步骤中制备
  7. 将肺细胞用冷PBS洗涤两次,并通过在450×g下旋转5分钟收集。
  8. 然后,将200-500μl细胞质提取物缓冲液加蛋白酶抑制剂加入到细胞沉淀中。
  9. 将沉淀轻轻混合,在冰上保持5分钟,然后以3,000xg离心5分钟。
  10. 收集上清液作为细胞质提取物。 通常,最终蛋白质浓度为约2.5-4μg/μl。
  11. 将沉淀用500μl细胞质提取缓冲液洗涤一次,悬浮于100-200μl核提取缓冲液中,在冰上轻轻混合5分钟,并以14,000×g离心5分钟。
  12. 收集上清液作为核提取物。 我们通常通过使用针对HDAC-1或组蛋白3作为核对照的抗体和作为细胞质蛋白质对照的Betta肌动蛋白的抗体进行蛋白质印迹来监测提取的蛋白质的纯度。

食谱

  1. 细胞质提取物缓冲液
    10 mM KCl
    10mM Hepes(pH7.9) 3mM MgCl 2/
    1.0%NP-40
  2. 核提取缓冲液
    400 mM KCl
    10mM Hepes(pH7.9) 3mM MgCl 2/
    1.0%NP-40

致谢

我们感谢Frederick国家癌症研究实验室的Yinling Hu,Robert H. Wiltrout和Jami Willette-Brown对实验设计的支持和建议。 这项工作由国家癌症研究所(ZIA BC 011212和ZIA BC 011391至Y.H.)支持。

参考文献

  1. Xiao,Z.,Jiang,Q.,Willette-Brown,J.,Xi,S.,Zhu,F.,Burkett,S.,Back,T.,Song,NY,Datla,M.,Sun, ,Goldszmid,R.,Lin,F.,Cohoon,T.,Pike,K.,Wu,X.,Schrump,DS,Wong,KK,Young,HA,Trinchieri,G.,Wiltrout,RH和Hu,Y 。(2013)。 IKKalpha在自发性肺鳞状细胞癌的发展中的关键作用 Cancer Cells 23(4):527-540。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Xiao, Z. and Jiang, Q. (2014). Protocol for Preparation of Nuclear Protein from Mouse Lungs. Bio-protocol 4(11): e1143. DOI: 10.21769/BioProtoc.1143.
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