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Nanofluidic Proteomic Immunoassay
纳流体蛋白质组免疫分析   

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Abstract

Nanofluidic proteomic immunoassay (NIA), consisting of isoelectric focusing followed by sensitive chemiluminescence detection, has the potential to quantitatively characterize protein post-translational modifications that cause shifts in isoelectric point (pI). This protocol details the NIA analysis of protein phosphorylation using AKT as an example.
This protocol can be used for two platforms, NanoPro 1000 and Peggy Sue, from ProteinSimple Company. NanoPro 1000 separates proteins based on charge. Peggy Sue separates proteins based on either charge or size. The platforms can analyze up to 96 samples at a time with lysates from as few as 25 cells per assay. Detailed information of the platforms is available on ProteinSimple’s website (http://www.proteinsimple.com).

Keywords: Immunoassay(免疫分析法), Antibody(抗体), AKT(Akt), NIA(NIA), Phosphorylation(磷酸化)

Materials and Reagents

  1. Reagents from ProteinSimple
    1. 25x aqueous protease/phosphatase inhibitor mix (ProteinSimple, catalog number: 040482 )
    2. 50x DMSO protease/phosphatase inhibitor mix (ProteinSimple, catalog number: 040510 )
    3. Premix G2 (Servalyt pH 5-8 separation gradient) (ProteinSimple, catalog number: 040974 )
    4. Premix G2 (pH 5-6 separation gradient) (ProteinSimple, catalog number: 040971 )
    5. PI standard 5.5 (ProteinSimple, catalog number: 040028 )
    6. PI standard ladder 3 (ProteinSimple, catalog number: 040646 )
    7. Anolyte refill (ProteinSimple, catalog number: 040337 )
    8. Catholyte refill (ProteinSimple, catalog number: 040338 )
    9. Wash concentrate refill (ProteinSimple, catalog number: 041108 )
    10. Amplified rabbit secondary antibody detection kit (ProteinSimple, catalog number: 041126 )
    11. Peroxide XDR (ProteinSimple, catalog number: 041084 )
    12.  Amplified rabbit secondary antibody detection kit (includes SA-HRP conjugate, secondary goat-anti-rabbit-biotin conjugate and antibody diluent) (ProteinSimple, catalog number: 041126 )
    13. RIPA lysis buffer (ProteinSimple, catalog number: 040483 ) (see Recipes)
    14. Bicine/CHAPS lysis buffer and sample diluent (ProteinSimple, catalog number: 040764 ) (see Recipes)
    15. Anolyte (10 mM Phosphoric Acid) (see Recipes)
    16. Catholyte (100 mM Sodium Hydroxide) (see Recipes)

  2. Antibodies for AKT analysis
    1. AKT (pan) antibody (Cell Signaling Technology, catalog number: 4691 )
    2. AKT1 antibody (BD Biosciences, catalog number: 610861 )
    3. AKT2 antibody (Cell Signaling Technology, catalog number: 3063 )
    4. Phospho-AKT (Thr308) (Cell Signaling Technology, catalog number: 2965 )
    5. Phospho-AKT (Thr450) (Cell Signaling Technology, catalog number: 9267 )
    6. Phospho-AKT (Ser473) (Cell Signaling Technology, catalog number: 9271 )

  3. Other materials
    1. BCA protein assay kit (Pierce Antibodies, catalog number: 23225 )
    2.  Dulbecco’s Modification of Eagle’s Medium (DMEM) with 4.5 g/L glucose & L-glutamine without pyruvate (Mediatech, catalog number: 10017CV )
    3. McCoy’s 5A (1x, with L-glutamine) (Mediatech, catalog number: 10050CV )
    4. Fetal bovine serum (FBS) (Life Technologies, Gibco®, catalog number: 10438026 )

Equipment

  1. NP1000 or Peggy (ProteinSimple, model: 004800 )
  2. Centrifuge for microplates (Thermo Fisher Scientific, model: Sorvall Legend XTR )
  3. Capillaries Charge Separation (ProteinSimple, catalog number: CBS701 )
  4. Assay Plate/Lid Kit (ProteinSimple, catalog number: 040663 )
  5. Sponge Pack (ProteinSimple, catalog number: 041528 )
  6. Tissue culture dish (100 mm) (Corning Incorporated, catalog number: 430167 )
  7. Microcentrifuge tube (Denville Scientific, catalog number: C2170 )

Software

  1. Compass version 2.5.11 (ProteinSimple)

Procedure

  1. Cell culture and lysate preparation
    1. Culture cells (such as HeLa or HCT116 cells) in DMEM or McCoy’s 5A medium supplemented with 10% FBS to 80-90% confluence in 10 cm plates. Treat cells as required.
    2. Gently wash cells in plates with 10 ml ice-cold PBS. Aspirate PBS thoroughly.
    3. Add 400 μl ice-cold RIPA or Bicine/CHAPS lysis buffer supplemented with both aqueous and DMSO protease/phosphatase inhibitor mix to each 10 cm plate on ice. Gently swirl plates to ensure good coverage and incubate for 10 min on ice.
    4. Scrape cells thoroughly. Pipet up and down to mix. Transfer buffer and cells to a 1.5 ml microfuge tube and incubate for additional 20 min on ice. Vortex the tube briefly every 5 min.
    5. Clarify by centrifugation (14,000 x g, 10 min) at 4 °C.
    6. Transfer supernatant to a fresh microfuge tube. Immediately aliquot supernatant (10-30 μl per aliquot) on ice and store at -80 °C.
    7. Measure protein concentrations using the BCA protein assay kit.
  2. Sample preparation for loading (for 12 lysates)
    1. In a microfuge tube, combine 110 µl premix G2 pH5-6, 110 µl premix G2 Servalyte pH5-8, 3.95 µl ladder 3, 1 µl Standard 5.5, and 4.5 µl DMSO protease/phosphatase inhibitor.
    2. Vortex for at least 15 sec to mix thoroughly. Aliquot 19 µl of the mix to each of 12 microfuge tubes.
    3. Dilute 5 μg of high concentration lysate with sample diluent or Bicine/CHAPS buffer to a final volume of 6 µl.
    4. Add the 6 µl of diluted lysate into one of the 12 tubes with 19 µl of the mix. The final protein concentration in capillary is 0.2 µg/µl.
    5. Add 10 µl of lysate mix into wells in row A (see Table 2).
  3. Primary antibody preparation
    1. Prepare primary antibodies at 1:50 dilution with antibody diluent (from the secondary antibody detection kit) in microfuge tubes.
    2. Add 10-20 μl of diluted primary antibodies into appropriate wells of 384-well assay plate (see Table 2).
  4. Secondary antibody preparation
    1. Prepare secondary antibodies at 1:100 dilution with antibody diluent in microfuge tubes.
    2. Add 10-20 μl of diluted secondary antibodies into appropriate wells of 384-well assay plate (see Table 2).
  5. Streptavidin-HRP preparation
    1. Prepare SA-HRP at 1:100 dilution with antibody diluent.
    2. Add 10-20 μl of diluted SA-HRP into appropriate wells of 384-well assay plate (see Table 2).
  6. Peroxide/Luminol preparation
    Combine 120 µl XDR peroxide and 120 µl luminol, mix well, and then add 10-20 μl of the mix into appropriate wells of the assay plate (see Table 2).
  7. Centrifuge the assay plate at 2,500 x g for 5 min to spin down the liquid and eliminate bubbles. Protect the plate from light before loading into the equipment.
  8. While the plate is spinning, create the assay file in Compass software. Set up as below for AKT assay.

    Table 1. Parameters of instrument settings
    Instrument setting
    Parameters
    Sample loading time
    25 sec
    Separation conditions
    40,000 μW, 40 min
    UV immobilization time
    60 sec
    Wash 1
    2 washes, 150 sec each (default)
    Primary antibody incubation
    120 min (default)
    Wash 2
    2 washes, 150 sec each (default)
    Secondary antibody incubation
    60 min (default)
    Wash 3
    2 washes, 150 sec each (default)
    Chemiluminescence exposure time
    30, 60, 120, 240 and 480 sec

    Table 2. An e
    xample of an assay layout. Assays are set up in 384-well plates. Table 2 was an example of an assay layout for 12 samples and 6 antibodies. Lysates were added into wells in row A from A1 to A12, respectively. Diluted primary antibodies were added into wells in row B (B1 to B12) to row G. Diluted secondary antibodies were added in row H (H1 to H12). Streptavidin-HRP was added into row I (I1 to I12). To avoid possible contamination between streptavidin-HRP and detection reagents in the plate, Lumino/Peroxide XDR was added into row A (A13 to A24) away from streptavidin-HRP.


    1. Start the run
      Launch the Start Run Wizard:
      1. Empty waste bottle and fill wash bottle with fresh ultrapure water.
      2. Replace sponge.
      3. Fill the wash buffer, Anolyte and Catholyte cups.
      4. Put capillary box in the primary capillary box position.
      5. Load sample plate.
      6. Click the “RUN” button to start the assay.
    2. Data analysis
      Data analysis is performed with the Compass software provided by ProteinSimple.
      1. In Image Analysis Settings, view data for different exposures in the Analysis screen. Choose the best exposure time according to the intensity of the signals.
      2. In Peak Fit Analysis Setting, set up Range (for AKT): minimum: 4.5, Maximum: 7.0.
      3. In Standard Analysis Settings, choose Std. Ladder 3 (premix pI 5-8) and add Standard 5.5 to the pI column and enter 500 in the Position column of the Fluorescent Peaks table. Select 4.9 and 7.0 for capillary registration by clicking the checkbox in the Registration column.

Representative data

  1. Representative results of the assay:
    Multiple peaks of AKT were detected by NIA using total AKT antibody representing both AKT1 and AKT2 isoforms as well as differences in post-translational modification (PTM) of each molecule (Fig. 1). No AKT3 is expressed in HCT116 cells.


    Figure 1. AKT isoforms characterized by NIA. Wt, AKT2-/-, AKT1-/-, and AKT1, 2 double knockout HCT116 cells were serum-starved overnight and lysed in NIA RIPA buffer. Samples were analyzed with anti-total AKT antibody. pI is shown on x axis and chemiluminescence on y axis.

Notes

  1. Keep samples on ice all the time.
  2. Always use protease and phosphatase inhibitors to preserve phosphorylation signals.
  3. Try to maintain low sample ionic strength to avoid shifts during IEF separation. The final total salt concentration should not exceed 50 mM in the capillary. Bicine/CHAPS or RIPA lysis buffers from ProteinSimple are recommended. It is imperative to obtain a high protein concentration if cells are harvested in RIPA buffer.
  4. Capillaries are light and humidity sensitive. Protect capillaries in opened packs from light and humidity.

Recipes

  1. RIPA lysis buffer
    20 mM HEPES (pH 7.5)
    150 mM NaCl
    1 % NP40 alternative
    0.25 % sodium deoxycholate
  2. Bicine/Chaps lysis buffer
    20 mM bicine (pH 7.6)
    0.6% CHAPS
  3. Anolyte (10 mM phosphoric acid)
  4. Catholyte (100 mM sodium hydroxide)

Acknowledgments

The work was supported by National Institutes of Health (NIH) grant 5R21CA126700 and a grant from the University of Texas MD Anderson Cancer Center Kidney Cancer Multidisciplinary Research Program to ZD. The protocol was adapted from our published paper in Oncogene (Guo et al., 2014) with modifications.

References

  1. Compass Software for NanoPro 1000 User Guide. (P/N 001-506, Revision 2, June 2013)
  2. Guo, H., Gao, M., Lu, Y., Liang, J., Lorenzi, P. L., Bai, S., Hawke, D. H., Li, J., Dogruluk, T., Scott, K. L., Jonasch, E., Mills, G. B. and Ding, Z. (2014). Coordinate phosphorylation of multiple residues on single AKT1 and AKT2 molecules. Oncogene 33(26): 3463-3472.
  3. ProteinSimple Nanopro Assay Development Guide. (P/N 041-015, Revision 3a, July 2011)

简介

纳米流体蛋白质组学免疫测定(NIA),由等电聚焦跟随敏感化学发光检测组成,具有定量表征导致等电点(pI)移位的蛋白质翻译后修饰的潜力。 该协议详细描述了使用AKT作为实例的蛋白质磷酸化的NIA分析。
此协议可用于ProteinSimple公司的两个平台NanoPro 1000和Peggy Sue。 NanoPro 1000基于电荷分离蛋白质。 Peggy Sue基于电荷或大小分离蛋白质。 平台可以一次分析高达96个样品,每个测定每裂解物少至25个细胞。 有关平台的详细信息,请访问ProteinSimple的网站( http://www.proteinsimple.com )。

关键字:免疫分析法, 抗体, Akt, NIA, 磷酸化

材料和试剂

  1. ProteinSimple的试剂
    1. 25x蛋白酶/磷酸酶抑制剂混合物(ProteinSimple,目录号:040482)
    2. 50x DMSO蛋白酶/磷酸酶抑制剂混合物(ProteinSimple,目录号:040510)
    3. 预混合物G2(Servalyt pH 5-8分离梯度)(ProteinSimple,目录号:040974)
    4. 预混料G2(pH 5-6分离梯度)(ProteinSimple,目录号:040971)
    5. PI标准5.5(ProteinSimple,目录号:040028)
    6. PI标准梯3(ProteinSimple,目录号:040646)
    7. 阳极电解液填充(ProteinSimple,目录号:040337)
    8. 阴极填充(ProteinSimple,目录号:040338)
    9. 洗涤浓缩液(ProteinSimple,目录号:041108)
    10. 扩增的兔二抗检测试剂盒(ProteinSimple,目录号:041126)
    11. 过氧化物XDR(ProteinSimple,目录号:041084)
    12.  扩增兔二抗检测试剂盒(包括SA-HRP 缀合物,第二山羊抗兔 - 生物素缀合物和抗体 稀释剂)(ProteinSimple,目录号:041126)
    13. RIPA裂解缓冲液(ProteinSimple,目录号:040483)(参见Recipes)
    14. Bicine/CHAPS裂解缓冲液和样品稀释液(ProteinSimple,目录号:040764)(参见配方)
    15. 阳极电解液(10 mM磷酸)(参见配方)
    16. 阴极电解液(100mM氢氧化钠)(见配方)

  2. 用于AKT分析的抗体
    1. AKT(pan)抗体(Cell Signaling Technology,目录号:4691)
    2. AKT1抗体(BD Biosciences,目录号:610861)
    3. AKT2抗体(Cell Signaling Technology,目录号:3063)
    4. 磷酸-AKT(Thr308)(Cell Signaling Technology,目录号:2965)
    5. 磷酸-AKT(Thr450)(Cell Signaling Technology,目录号:9267)
    6. 磷酸-AKT(Ser473)(Cell Signaling Technology,目录号:9271)
  3. 其他材料
    1. BCA蛋白测定试剂盒(Pierce Antibodies,目录号:23225)
    2. & Dulbecco's Modification of Eagle's Medium(DMEM)with 4.5g/L glucose & 没有丙酮酸的L-谷氨酰胺(Mediatech,目录号:10017CV)
    3. McCoy's 5A(1x,具有L-谷氨酰胺)(Mediatech,目录号:10050CV)
    4. 胎牛血清(FBS)(Life Technologies,Gibco ,目录号:10438026)

设备

  1. NP1000或Peggy(ProteinSimple,型号:004800)
  2. 用于微孔板的离心机(Thermo Fisher Scientific,型号:Sorvall Legend XTR)
  3. 毛细管电荷分离(ProteinSimple,目录号:CBS701)
  4. 测定板/盖试剂盒(ProteinSimple,目录号:040663)
  5. 海绵包(ProteinSimple,目录号:041528)
  6. 组织培养皿(100mm)(Corning Incorporated,目录号:430167)
  7. 微量离心管(Denville Scientific,目录号:C2170)

软件

  1. 指南针版本2.5.11(ProteinSimple)

程序

  1. 细胞培养和裂解物制备
    1. 培养细胞(例如HeLa或HCT116细胞)在DMEM或McCoy's 5A中 培养基补充10%FBS至80-90%汇合在10cm平板中。 根据需要处理细胞。
    2. 轻轻洗涤板的细胞用10毫升冰冷PBS。 彻底吸出PBS。
    3. 加入400μl冰冷的RIPA或Bicine/CHAPS裂解缓冲液补充   水性和DMSO蛋白酶/磷酸酶抑制剂混合至每10cm 在冰上的板。 轻轻旋转板,以确保良好的覆盖和孵化 在冰上10分钟。
    4. 彻底刮细胞。 吸取上下 混合。 转移缓冲液和细胞到1.5毫升微量离心管和孵化 在冰上另外20分钟。 每5分钟涡旋管。
    5. 在4℃下通过离心(14,000×g,10分钟)澄清
    6. 转移上清液到新鲜微量离心管。 立即等分 上清液(每个等分试样10-30μl)并储存在-80℃
    7. 加入400μl冰冷的RIPA或Bicine/CHAPS裂解缓冲液补充   水性和DMSO蛋白酶/磷酸酶抑制剂混合至每10cm 在冰上的板。 轻轻旋转板,以确保良好的覆盖和孵化 在冰上10分钟。
    8. 彻底刮细胞。 吸取上下 混合。 转移缓冲液和细胞到1.5毫升微量离心管和孵化 在冰上另外20分钟。 每5分钟涡旋管。
    9. 在4℃下通过离心(14,000×g,10分钟)澄清
    10. 转移上清液到新鲜微量离心管。 立即等分 上清液(每个等分试样10-30μl)并储存在-80℃
    11. ... Add the 6 µl of diluted lysate into one of the 12 tubes with 19 µl of the mix. The final protein concentration in capillary is 0.2 µg/µl.
    12. Add 10 µl of lysate mix into wells in row A (see Table 2).
  2. Primary antibody preparation
    1. Prepare primary antibodies at 1:50 dilution with antibody diluent (from the secondary antibody detection kit) in microfuge tubes.
    2. Add 10-20 μl of diluted primary antibodies into appropriate wells of 384-well assay plate (see Table 2).
  3. Secondary antibody preparation
    1. Prepare secondary antibodies at 1:100 dilution with antibody diluent in microfuge tubes.
    2. Add 10-20 μl of diluted secondary antibodies into appropriate wells of 384-well assay plate (see Table 2).
  4. Streptavidin-HRP preparation
    1. Prepare SA-HRP at 1:100 dilution with antibody diluent.
    2. 将10-20μl稀释的SA-HRP加入384孔测定板的适当孔中(见表2)。
  5. 过氧化物/鲁米诺制剂
    将120μlXDR过氧化物和120μl鲁米诺混合均匀,然后将10-20μl混合物加入测定板的适当孔中(见表2)。
  6. 以2,500×g离心测定板5分钟以旋转液体并除去气泡。 在装入设备之前,请保护平板免受光照。
  7. 在板旋转时,在Compass软件中创建测定文件。 设置如下,用于AKT测定。

    表1.仪器设置的参数
    仪器设置
    参数
    样品装载时间
    25秒
    分离条件
    40,000μW,40分钟
    紫外线固定时间
    60秒
    洗涤1
    2次洗涤,每次150秒(默认)
    第一抗体孵育
    120分钟(默认)
    清洗2
    2次洗涤,每次150秒(默认)
    二抗孵育
    60分钟(默认)
    洗涤3
    2次洗涤,每次150秒(默认)
    化学发光曝光时间
    30,60,120,240和480秒

    表2.测定布局的 样品 。在384孔板中设置测定。 表2是一个例子 12个样品和6个抗体的测定布局。 加入裂解物 A行中的孔分别为A1至A12。 稀释的一抗 加入到B行(B1至B12)至G行的孔中 在H行(H1至H12)中加入抗体。 加入链霉亲和素-HRP 到行I(I1到I12)。 以避免可能的污染 链霉亲和素-HRP和板中的检测试剂,Lumino /过氧化物 将XDR加入到A链(A13至A24)中远离链霉抗生物素蛋白-HRP

    1. 开始运行
      启动启动运行向导:
      1. 清空废液瓶,并用新鲜超纯水填充洗瓶
      2. 更换海绵。
      3. 填充洗涤缓冲液,Anolyte和Catholyte杯。
      4. 将毛细管盒放在主毛细管盒的位置。
      5. 装载样品板。
      6. 点击"运行"按钮开始测定。
    2. 数据分析
      用ProteinSimple提供的Compass软件进行数据分析。
      1. 在"图像分析设置"中,查看不同曝光的数据 分析"屏幕。 根据选择最佳曝光时间 信号强度。
      2. 在峰值拟合分析设置中,设置范围(对于AKT):最小值:4.5,最大值:7.0
      3. 在标准分析设置中,选择标准。 梯子3(预混合pI 5-8),并将标准5.5添加到pI列,并在位置中输入500   列的荧光峰。 毛细管选择4.9和7.0   通过单击注册列中的复选框进行注册。

代表数据

  1. 测定的代表性结果:
    通过NIA使用代表AKT1和AKT2同种型的总AKT抗体以及每个分子的翻译后修饰(PTM)的差异检测AKT的多个峰(图1)。 在HCT116细胞中没有AKT3表达

    图1.以NIA为特征的AKT同种型。 Wt,AKT2 -/- ,AKT1 -/- 和AKT1,2双敲除HCT116细胞血清饥饿过夜,并在NIA RIPA缓冲液中裂解。 用抗总AKT抗体分析样品。 pI显示在x轴上,化学发光显示在y轴上

笔记

  1. 始终将样品保持在冰上。
  2. 始终使用蛋白酶和磷酸酶抑制剂来保存磷酸化信号
  3. 尝试保持低样品离子强度,以避免在IEF分离期间的偏移。 毛细管中的最终总盐浓度不应超过50mM。 推荐使用来自ProteinSimple的Bicine/CHAPS或RIPA裂解缓冲液。 如果细胞在RIPA缓冲液中收获,则必须获得高蛋白浓度。
  4. 毛细管对光和湿度敏感。 保护打开包装中的毛细管免受光照和湿度的影响

食谱

  1. RIPA裂解缓冲液
    20mM HEPES(pH7.5) 150mM NaCl 1%NP40替代品
    0.25%脱氧胆酸钠
  2. Bicine/Chaps裂解缓冲液
    20mM N-二(羟乙基)甘氨酸(pH7.6) 0.6%CHAPS
  3. 阳极电解液(10mM磷酸)
  4. 阴极电解液(100mM氢氧化钠)

致谢

这项工作是由国家卫生研究院(NIH)拨款5R21CA126700和德克萨斯大学MD安德森癌症中心肾癌多学科研究计划授予ZD的资助。 该方案改编自我们在Oncogene(Guo等人,2014)中发表的论文,并进行了修改。

参考文献

  1. 指南针软件,用于 NanoPro 1000用户指南。 (P/N 001-506,修订版2,2013年6月)
  2. Guo,H.,Gao,M.,Lu,Y.,Liang,J.,Lorenzi,PL,Bai,S.,Hawke,DH,Li,J.,Dogruluk,T.,Scott,KL,Jonasch,E 。,Mills,GB和Ding,Z.(2014)。 协调单个AKT1和AKT2分子上多个残基的磷酸化。癌基因 33(26):3463-3472。
  3. ProteinSimple Nanopro Assay开发指南( P/N 041-015,Revision 3a,2011年7月)
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引用:Guo, H., Lorenzi, P. L. and Ding, Z. (2015). Nanofluidic Proteomic Immunoassay. Bio-protocol 5(14): e1537. DOI: 10.21769/BioProtoc.1537.
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