欢迎您, 登录 | 注册

首页 | English

X
加载中

Pulse-chase method is a powerful technique used to follow the dynamics of proteins over a period of time. The expression level, processing, transport, secretion or half-life of proteins can be tracked by metabolically labeling the cells, such as with radiolabeled amino acids (pulse step). This protocol describes the condition used to study the folding and disulfide bond formation of immunoglobulin in suspension cells. With some minor modifications, this protocol can be adapted to study the degradation rate or the secretion of target proteins.

Thanks for your further question/comment. It has been sent to the author(s) of this protocol. You will receive a notification once your question/comment is addressed again by the author(s).
Meanwhile, it would be great if you could help us to spread the word about Bio-protocol.

X

Pulse Chase of Suspension Cells
悬浮细胞的脉冲追踪

免疫学 > 抗体分析 > 抗体检测
作者: Lai-Yee Wong
Lai-Yee WongAffiliation: Department of Molecular Microbiology and Immunology, University of Southern California, Los Angeles, CA, USA
For correspondence: laiyee.wong@usc.edu
Bio-protocol author page: a1453
QiMing Liang
QiMing LiangAffiliation: Department of Molecular Microbiology and Immunology, University of Southern California, Los Angeles, CA, USA
Bio-protocol author page: a1454
Kevin Brulois
Kevin BruloisAffiliation: Department of Molecular Microbiology and Immunology, University of Southern California, Los Angeles, CA, USA
Bio-protocol author page: a1455
 and Jae Jung
Jae JungAffiliation: Department of Molecular Microbiology and Immunology, University of Southern California, Los Angeles, CA, USA
Bio-protocol author page: a1456
Vol 4, Iss 13, 7/5/2014, 3631 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1170

[Abstract] Pulse-chase method is a powerful technique used to follow the dynamics of proteins over a period of time. The expression level, processing, transport, secretion or half-life of proteins can be tracked by metabolically labeling the cells, such as with radiolabeled amino acids (pulse step). This protocol describes the condition used to study the folding and disulfide bond formation of immunoglobulin in suspension cells. With some minor modifications, this protocol can be adapted to study the degradation rate or the secretion of target proteins.
Keywords: Protein folding(蛋白质折叠), Protein degradation(蛋白质的降解), Protein secretion(分泌蛋白)

[Abstract]

Materials and Reagents

  1. Pulse chase
    1. Cells growing in suspension
    2. HBSS (Life Technologies, InvitrogenTM, catalog number: 14175-095 )
    3. RPMI without methionine and cysteine (Sigma-Aldrich, catalog number: R7513 )
    4. Dialyzed FBS (Life Technologies, InvitrogenTM, catalog number: 26400044 )
    5. N-Ethylmaleimide (NEM) (Sigma-Aldrich, catalog number: R3876 )
    6. Cyclohexamide (CHX) (Sigma-Aldrich, catalog number: C7698 )
    7. Express 35S protein labelling mix (Perkin Elmer, catalog number: NEG072014MC )
    8. Methionine (Sigma-Aldrich, catalog number: M5308 )
    9. Cysteine (Sigma-Aldrich, catalog number: C7352 )
    10. Labeling medium (see Recipes)
    11. Chase medium (see Recipes)
    12. 2x stop buffer (see Recipes)

  2. Cell lysis and immunoprecipitation
    1. Antibody against protein of interest
    2. Protein A/G beads (Thermo Fisher Scientific, catalog number: 20422 )
    3. Complete Protease Inhibitor Tablets (Roche Diagnostics, catalog number: 11836145001 )
    4. Lysis buffer (see Recipes)

  3. SDS-PAGE
    1. 4-12% Bis-Tris protein gel (Life Technologies, InvitrogenTM)
    2. MOPS running buffer (Life Technologies, InvitrogenTM, catalog number: NP0001 )
    3. Amplify solution (GE Healthcare, catalog number: NAMP100 )
    4. Gel drying solution (Life Technologies, InvitrogenTM, catalog number: LC4025 )
    5. Gel fixing solution (see Recipes)

Equipment

  1. Incubator
  2. Eppendorf tube
  3. 26-gauge needle
  4. 1 ml syringe
  5. Heat block
  6. Gel dryer
  7. Phosphor imaging screen

Procedure

Cells are pulse-labeled and chased in a single tube and an aliquot of cells is removed from this tube for each time point of the chase.
After determining the numbers of chase time points (x), prepare enough cells for the experiment (x + 1, 2 x 106 per sample). See Note 1.

  1. Pulse chase
    1. Wash cells (2 x 106 per sample) with 2 ml of HBSS.
    2. Pellet cells at 500 x g for 3 min at room temperature. Resuspend cells in 2 ml/sample of pre-warmed labeling medium. Mix gently.
    3. Incubate the cells for 20 min at 37 ℃ incubator.
    4. Pellet cells at 500 x g for 3 min at room temperature and resuspend cells in 100 μl/sample of pre-warmed labeling medium. Keep the cells at 37 ℃, either in a water bath or an incubator during the labeling and chase periods.
    5. Pulse for 2 min at 37 ℃ with [35S] methionine (100 μCi/ml) (see Note 2).
    6. Add 400 μl/sample of chase medium. Pipet up and down gently to ensure proper mixing.
    7. Immediately take out 500 μl for the 0 min sample. Transfer to an eppendorf tube filled with 500 μl of 2x ice cold stop buffer (see Note 3).
    8. Spin cells at 500 x g for 2 min at 4 ℃ and freeze pellet.
    9. Repeat steps 7-8 for every time point.
    10. Proceed to cell lysis or keep the pellet frozen in -80 ℃.

  2. Cell lysis and immunoprecipitation
    1. Add 1ml cold lysis buffer to each cell pellet.
    2. Apply mechanical shearing force to the cell lysate by passing it through a 26-gauge needle attached to a 1 ml syringe (repeat 5-10 times for thorough lysis). Incubate on ice for 15 min. Spin at 16,000 x g for 15 min at 4 ℃ to clarify the lysate. Transfer the clarified cell extracts to a new tube.
    3. Add antibody against protein of interest to equal volume of cell extracts and rotate overnight at 4 ℃ (see Note 4).
    4. Add 30 μl of Protein A/G beads and incubate for another 2 h at 4 ℃.
    5. Wash the immunoprecipitates twice with 1 ml of lysis buffer.
    6. Add 50 μl of sample buffer without reducing agents (such as DTT or 2-mercaptoenthanol). See Note 5.
    7. Heat the samples 65 °C for 5 min. Centrifuge briefly before proceeding to non-reducing SDS-PAGE.

  3. SDS-PAGE
    1. Load 25 μl of the immunoprecipitates on SDS-polyacrylamide gel.
    2. Immerse the gel in fixing buffer for 15-30 min at room temperature.
    3. Immerse the gel in amplify solution for 15 min at room temperature.
    4. Immerse the gel in gel drying solution for another 15 min at room temperature.
    5. Dry the gel on filter paper on top of a gel dryer of choice.
    6. [35S] methionine-labeled proteins can be visualized after exposure to a Phosphor imaging screen.

Representative data

  1. The protocol described here was used to examine the maturation kinetics of pentameric IgM complexes during their passage through the secretory pathway. IgM assembly begins with the coupling of a heavy chain (H) and a light chain (L), resulting in a monomeric heavy and light chain intermediate (HL). A H2L2 unit is assembled and followed by large multimers of “H2L2”. At later points of the chase time, the signals for these IgM intermediates, especially the high molecular weight species, will decrease due to the successful assembly and secretion of mature IgM.


    Figure 1. I.29 μ+ mouse lymphoma cells were pulse labeled with 35S-methoninie for 2 min and chased for the indicated times. The cell extracts were immunoprecipitated with αIgM and the immunoprecipitates were resolved by non-reducing SDS/PAGE. IgM assembly intermediates are indicated.

Notes

  1. In order to ensure equal number of cells are used in each samples, it is necessary to determine the number of chase time points (x) and the volume of chase medium before starting the experiment. We normally prepare enough cells and medium for x (number of chase time points) plus 1 to account for any fluid loss during the experiment.
  2. The incubation time with radioactive amino acids should be optimized depending on the target protein and the cells used. Subsequently, keeping the labeling step consistent, especially the number of cells and ‘pulse’ time with radioactive amino acids, will reduce variability in the end results.
  3. Alternatively, the cells can be added directly to ice cold 2x lysis buffer and cell lysis can commence immediately.
  4. The amount of antibody and cell extracts to be added and the incubation time for antibody-antigen binding should be determined and optimized according to each antibody and antigen. Incubation time can vary from 1 h to overnight at 4 ℃.
  5. Denaturing sample buffer with reducing agents can be used if disulfide bond formation is not being monitored.
  6. Dialyzed FBS is used to prevent contamination of ‘cold’ methionine and cysteine. The amount added to the medium should be determined according to the cell line used.
  7. Cyclohexamide (CHX) is added to reduce the level of newly translated protein. CHX as well as 'cold' methionine and cysteine should be added fresh to the medium.
  8. NEM is an alkylating agent that covalently attaches to free SH-groups found on cysteines. It is added to prevent disulfide bonds from forming once the chase period has ended. In pulse-chase experiments that track the formation of disulfide bonds, NEM must be added to the stop and lysis buffers.

Recipes

  1. Labeling medium
    RPMI lacking methionine or cysteine
    1% Penicilin and Streptomycin
    1% glutamine
    5-10% dialyzed FBS (see Note 6)
  2. Chase medium (see Note 7)
    Labeling medium (see above) plus
    5 mM cysteine
    5 mM methionine
    1 mM CHX
  3. 2x stop buffer
    HBSS with 40 mM NEM (see Note 8)
  4. Lysis buffer
    50 mM Tris (pH 7.4)
    150 mM NaCl
    0.5% NP-40
    0.5% Na deoxycholate
    20 mM NEM
    Protease inhibitors
  5. Gel fixing solution
    25% methanol
    15% acetic acid

Acknowledgments

The protocol presented here was adapted from van Anken et al. (2009). This work was partly supported by grants from the National Institutes of Health (CA082057, CA31363, CA115284, CA180779, AI105809, and AI073099); the Hastings Foundation; the Fletcher Jones Foundation and BaCaTec (J.U.J.). We also thank the Jung laboratory members for their support and discussions.

Reference

  1. van Anken, E., Pena, F., Hafkemeijer, N., Christis, C., Romijn, E. P., Grauschopf, U., Oorschot, V. M., Pertel, T., Engels, S., Ora, A., Lastun, V., Glockshuber, R., Klumperman, J., Heck, A. J., Luban, J. and Braakman, I. (2009). Efficient IgM assembly and secretion require the plasma cell induced endoplasmic reticulum protein pERp1. Proc Natl Acad Sci U S A 106(40): 17019-17024.
  2. Wong L. Y., Brulois K., Toth Z., Inn K. S., Lee S. H., O'Brien K., Lee H., Gao S. J., Cesarman E., Ensser A. and Jung J. U. (2013). The product of Kaposi's sarcoma-associated herpesvirus immediate early gene K4.2 regulates immunoglobulin secretion and calcium homeostasis by interacting with and inhibiting pERP1. J Virol 87(22): 12069-12079.

材料和试剂

  1. 脉冲追逐
    1. 悬浮在悬浮液中的细胞
    2. HBSS(Life Technologies,Invitrogen TM ,目录号:14175-095)
    3. 无甲硫氨酸和半胱氨酸的RPMI(Sigma-Aldrich,目录号:R7513)
    4. 透析的FBS(Life Technologies,Invitrogen TM ,目录号:26400044)
    5. (NEM)(Sigma-Aldrich,目录号:R3876)。
    6. 环己酰胺(CHX)(Sigma-Aldrich,目录号:C7698)
    7. Express S蛋白标记混合物(Perkin Elmer,目录号:NEG072014MC)
    8. 甲硫氨酸(Sigma-Aldrich,目录号:M5308)
    9. 半胱氨酸(Sigma-Aldrich,目录号:C7352)
    10. 标签介质(参见配方)
    11. Chase介质(见配方)
    12. 2x停止缓冲区(参见配方)

  2. 细胞裂解和免疫沉淀
    1. 抗感兴趣的蛋白质的抗体
    2. 蛋白A/G珠(Thermo Fisher Scientific,目录号:20422)
    3. 完全蛋白酶抑制剂片剂(Roche Diagnostics,目录号:11836145001)
    4. 裂解缓冲液(见配方)

  3. SDS-PAGE
    1. 4-12%Bis-Tris蛋白凝胶(Life Technologies,Invitrogen )
    2. MOPS运行缓冲液(Life Technologies,Invitrogen TM ,目录号:NP0001)
    3. 扩增溶液(GE Healthcare,目录号:NAMP100)
    4. 凝胶干燥溶液(Life Technologies,Invitrogen TM,目录号:LC4025)
    5. 凝胶固定溶液(见配方)

设备

  1. 孵化器
  2. Eppendorf管
  3. 26号针
  4. 1 ml注射器
  5. 热块
  6. 凝胶干燥器
  7. 荧光成像屏

程序

在单个管中对细胞进行脉冲标记和追踪,并且对于追踪的每个时间点从该管中取出细胞的等分试样。
在确定追踪时间点(x)的数目后,为实验准备足够的细胞(x + 1,2x 10 6个样品)。 见注1。

  1. 脉冲追逐
    1. 用2ml HBSS洗涤细胞(每个样品2×10 6个)。
    2. 粒细胞 在500×g下在室温下温育3分钟。 重悬细胞在2毫升/样品   的预热标记培养基。 轻轻混匀。
    3. 孵育细胞在37℃培养箱中20分钟。
    4. 在室温下将沉淀细胞以500×g离心3分钟并重悬 细胞在100μl/样品的预热标记培养基中。 保持细胞 37℃,在标记过程中在水浴或培养箱中 追逐期。
    5. 在37℃下用[ S]甲硫氨酸(100μCi/ml)(参见注2)脉冲2分钟。
    6. 加入400μl/Chase培养基的样品。 轻轻地上下移动以确保正确混合。
    7. 立即取出500μl的0分钟样品。 转移到 Eppendorf管中装入500μl2×冰冷的终止缓冲液(见注释 3)。
    8. 在4℃下以500×g离心细胞2分钟,冷冻沉淀
    9. 对每个时间点重复步骤7-8。
    10. 继续细胞裂解或保持颗粒冷冻在-80℃

  2. 细胞裂解和免疫沉淀
    1. 向每个细胞沉淀中加入1ml冷裂解缓冲液
    2. 应用机械 剪切力通过使其通过26-规格的细胞裂解物 针连接到1ml注射器(重复5-10次彻底 裂解)。 在冰上孵育15分钟。 在4,000rpm下旋转16,000in x g 15分钟 ℃澄清裂解液。 将澄清的细胞提取物转移到新的 管
    3. 向等体积的细胞提取物中加入抗感兴趣的蛋白质的抗体,并在4℃下旋转过夜(参见注释4)
    4. 加入30微升蛋白A/G珠,并在4℃下再孵育2小时
    5. 用1ml裂解缓冲液洗涤免疫沉淀两次
    6. 加入50μl无还原剂(如DTT或2-巯基乙醇)的样品缓冲液。 见注5.
    7. 将样品在65℃加热5分钟。 在进行非还原SDS-PAGE之前简单离心。

  3. SDS-PAGE
    1. 加载25微升免疫沉淀在SDS-聚丙烯酰胺凝胶上
    2. 将凝胶在室温下浸入固定缓冲液中15-30分钟
    3. 将凝胶在室温下在扩增溶液中浸泡15分钟
    4. 将凝胶在凝胶干燥溶液中在室温下浸泡15分钟
    5. 在选择的凝胶干燥器顶部的滤纸上干燥凝胶。
    6. 在暴露于荧光成像屏幕后,可以显现[35 S]甲硫氨酸标记的蛋白质。

代表数据

  1. 本文所述的方案用于检查五聚体IgM复合物在通过分泌途径途径期间的成熟动力学。 IgM组装开始于重链(H)和轻链(L)的偶联,产生单体重链和轻链中间体(HL)。 组装H2L2单元,随后是"H2L2"的大多聚体。 在追踪时间的后面的点,由于成功的IgM的成功组装和分泌,这些IgM中间体,特别是高分子量物质的信号将减少。


    图1. I.29μ + 小鼠淋巴瘤细胞用 S-methoninie 2分钟,并按指定时间追踪。 细胞提取物用αIgM免疫沉淀,免疫沉淀物通过非还原SDS/PAGE分离。指示IgM装配中间体

笔记

  1. 为了确保每个样品中使用相等数量的细胞,必须在开始实验之前确定追踪时间点(x)的数量和追踪培养基的量。我们通常准备足够的细胞和介质为x(追逐时间点的数量)加1,以解释实验期间的任何液体损失。
  2. 与放射性氨基酸的孵育时间应根据目标蛋白和使用的细胞进行优化。随后,保持标记步骤一致,特别是细胞数量和用放射性氨基酸的"脉冲"时间,将减少最终结果的变异性。
  3. 或者,可以将细胞直接加入到冰冷的2x裂解缓冲液中,并且可以立即开始细胞裂解。
  4. 应当根据每种抗体和抗原确定和优化待添加的抗体和细胞提取物的量以及抗体 - 抗原结合的孵育时间。孵育时间可以在4℃下从1小时到过夜不等。
  5. 如果不监测二硫键形成,可以使用具有还原剂的变性样品缓冲液
  6. 透析的FBS用于防止"冷"甲硫氨酸和半胱氨酸的污染。添加到培养基中的量应根据所用的细胞系确定。
  7. 加入环己酰胺(CHX)以降低新翻译的蛋白质的水平。 CHX以及"冷"甲硫氨酸和半胱氨酸应该新鲜加入到培养基中
  8. NEM是共价连接到在半胱氨酸上发现的游离SH-基团的烷化剂。加入它以防止追赶期结束后形成二硫键。在追踪二硫键形成的脉冲追踪实验中,必须向停止和裂解缓冲液中加入NEM。

食谱

  1. 标记媒体
    缺乏甲硫氨酸或半胱氨酸的RPMI 1%青霉素和链霉素 1%谷氨酰胺 5-10%透析的FBS(参见注释6)
  2. Chase介质(见注7)
    标签介质(见上文)加上
    5mM半胱氨酸 5mM甲硫氨酸 1 mM CHX
  3. 2x停止缓冲区
    HBSS与40mM NEM(参见注释8)
  4. 裂解缓冲液
    50mM Tris(pH7.4) 150mM NaCl 0.5%NP-40
    0.5%脱氧胆酸钠 20mM NEM
    蛋白酶抑制剂
  5. 凝胶定影液
    25%甲醇
    15%乙酸

致谢

本文提供的方案改编自van Anken等人(2009)。这项工作部分得到国立卫生研究院(CA082057,CA31363,CA115284,CA180779,AI105809和AI073099)的赠款支持;黑斯廷斯基金会; Fletcher Jones基金会和BaCaTec(J.U.J.)。我们还感谢Jung实验室成员的支持和讨论。

参考

  1. van Anken,E.,Pena,F.,Hafkemeijer,N.,Christis,C.,Romijn,EP,Grauschopf,U.,Oorschot,VM,Pertel,T.,Engels,S.,Ora, ,V.,Glockshuber,R.,Klumperman,J.,Heck,AJ,Luban,J。和Braakman,I。(2009)。 有效的IgM装配和分泌需要浆细胞诱导的内质网蛋白pERp1。 Proc Natl Acad Sci USA 106(40):17019-17024。
  2. Wong L.Y.,Brulois K.,Toth Z.,Inn K.S.,Lee S.H.,O'Brien K.,Lee H.,Gao S.J.,Cesarman E.,Ensser A.and Jung J.U。(2013)。 卡波西肉瘤相关疱疹病毒立即早期基因K4.2的产物调节免疫球蛋白分泌和钙稳态与pERP1相互作用并抑制pERP1。 Virot 87(22):12069-12079。
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。

X


How to cite this protocol: Wong, L., Liang, Q., Brulois, K. and Jung, J. (2014). Pulse Chase of Suspension Cells. Bio-protocol 4(13): e1170. DOI: 10.21769/BioProtoc.1170; Full Text



可重复性反馈:

  • 添加图片
  • 添加视频

我们的目标是让重复别人的实验变得更轻松,如果您已经使用过本实验方案,欢迎您做出评价。我们鼓励上传实验图片或视频与小伙伴们(同行)分享您的实验心得和经验。(评论前请登录)

问题&解答:

  • 添加图片
  • 添加视频

(提问前,请先登陆)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或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。


登陆 | 注册
引用格式
分享
Twitter Twitter
LinkedIn LinkedIn
Google+ Google+
Facebook Facebook