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Detection of Phospho-KRAS by Electrophoretic Mobility Change in Human Cell Lines and in Tumor Samples from Nude Mice Grafts
利用电泳迁移率的变化检测人源细胞系和裸小鼠移植肿瘤样本中的KRAS磷酸化   

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参见作者原研究论文

本实验方案简略版
Cancer Research
Feb 2014

Abstract

KRAS is the oncogene most frequently mutated in human solid tumors especially in pancreas, colon, small intestine, biliary tract and lung. We have recently demonstrated that oncogenic KRAS needs S181 phosphorylation to fully display its oncogenic features suggesting its inhibition as a therapeutic treatment against KRAS-driven tumors. Due to the importance to detect KRAS phosphorylation in human tumors and the absence of specific antibodies against phosphorylated KRAS, we developed a new protocol based on the Phos-tag SDS methodology to detect this post-translational modification for KRAS. Phos-tag is a molecule that binds specifically to phosphorylated proteins, decreasing their migration speed in SDS-PAGE and allowing its separation from the non-phosphorylated forms.

Materials and Reagents

  1. Human tumor cell lines
  2. Human tumor samples grown in immunodeficient mice from living cells or tissue origins
  3. Phos-tagTM acrylamide (Wako Chemicals GmbH, catalog number: 304-93521 , #AAL-107)
  4. λ Protein phosphatase (≥400,000 units/ml) (Calbiochem, catalog number: 539514-20KV )
  5. Anti- c- KRAS (clone Ab-1) mouse mAb (Calbiochem, catalog number: OP24 ) or any antibody against a putative tag-KRAS expressed (HA tag has been tested successfully)
  6. Protease inhibitors final concentrations
    1. Aprotinin: 150 nM (1 μg/ml) (Sigma-Aldrich, catalog number: A1153 ) (recommended stock solution 1 mg/ml)
    2. Leupeptin: 20 μM (10 μg/ml) (Sigma-Aldrich, catalog number: L2884 ) (recommended stock solution 1 mg/ml)
    3. Phenylmethanesulfonyl fluoride (PMSF): 1 mM (Sigma-Aldrich, catalog number: S6508 ) (recommended stock solution 100 mM)
  7. Phosphatase inhibitors
    1. Sodium orthovanadate: 0.2 mM (Sigma-Aldrich, catalog number: S6508) (recommended stock solution 200 mM)
    2. Sodium fluoride: 5 mM (Sigma-Aldrich, catalog number: S7920 ) (recommended stock solution 1 M)
  8. IGEPAL CA-630 (Sigma-Aldrich, catalog number: I8896 )
  9. Dynabeads Protein G for immunoprecipitation (Novex by Life Technologies, catalog number: 10003D )
  10. Temed
  11. 13% ammonium persulfate (APS)
  12. MnCl2 stock solutions 10 mM and 25 mM
  13. λ phosphatase lysis buffer (see Recipes)
  14. 3x SDS sample buffer (see Recipes)
  15. Electrophoresis running buffer (see Recipes)
  16. Transfer buffer (see Recipes)
  17. 5 mM Phos-tag stock solution (see Recipes)
  18. 20x PBS (see Recipes)
  19. Solution 1 for SDS-PAGE (see Recipes)
  20. Solution 2 for SDS-PAGE (see Recipes)
  21. Solution 3 for SDS-PAGE (see Recipes)
  22. Resolving composition of a 12% SDS-PAGE mini gel (1.5 mm thickness) for Phos-tag (see Recipes)
  23. Stacking composition of a 12% SDS-PAGE mini gel (1.5 mm thickness) (see Recipes)

Equipment

  1. Tissue grinder (Dounce) (1 ml) (Wheaton, catalog number: 357538 )
  2. Magnet DynaMagTM-2 (Life Technologies, catalog number: 12321D )
  3. Tube Rotator (Bibby Scientific Ltd., Stuart SB2)

Procedure

  1. Sample lysis and λ Phosphatase treatment

    For all samples
    1. For each sample analysed (sample A), a negative control (sample B) treated with λ Phosphatase will be assayed concurrently. Prepare two tubes with ice-cold λ Phosphatase Lysis Buffer, freshly prepared, containing protease and phosphatase inhibitors (A) or only protease inhibitors (B).


      Figure 1. The flowchart of sample lysis and λ Phosphatase treatment

    For cell lines
    1. Wash cells from a 10 cm dish (80% confluence) with 10 ml ice-cold phosphate buffered saline (PBS) twice.
    2. Scrape cells with 1 ml ice-cold PBS, collect in two 1.5 ml microfuge tubes A and B (500 μl each) and spin for 3 min at 1,000 x g at 4 °C. Discard supernatant.
    3. Add 18 μl ice-cold λ phosphatase lysis buffer containing only protease inhibitors to the pellet B and next 2 μl of λ Protein Phosphatase (40 units/ μl final concentration). Incubate at 30 °C for 30 min in a dry block.
    4. Add 180 μl ice-cold λ Phosphatase Lysis Buffer containing protease and phosphatase inhibitors to the pellet A. Resuspend the pellet by pipetting and after 10 min on ice, spin for 10 min at 10,000 x g at 4 °C. Transfer the supernatant to a new 1.5 ml tube (sample lysate A).
    5. Add 160 μl cold λ phosphatase lysis buffer containing protease and phosphatase inhibitors to the pellet B. Resuspend the pellet and after 10 min on ice, spin for 10 min at 10,000 x g at 4 °C.Transfer the supernatant to a new 1.5 ml tube (sample lysate B).
    6. Analyse protein content of the supernatant obtained from steps 4-5 by Lowry method.

    For tumor samples
    1. Take two fragments of the same tumor of approximately 0.1 g each. Leave each fragment of tumor on a 35 mm dish on ice in the presence of 0.4 ml of cold λ phosphatase lysis buffer containing protease and phosphatase inhibitors (dish sample A) or only protease inhibitors (dish sample B), but not DTT and MnCl2 in any case (will be added in step 5). Mince the tumors in the smallest possible pieces with a scalpel and scissors and collect all volume, containing minced tissue and lysis buffer, in 1.5 ml microfuge tubes.
      Note: If the sample is a frozen tumor, mincing of the tumor in the presence of the buffer has to be done while the tumor is thawing.
    2. Homogenize the samples with a tissue grinder on ice until no clumps remain in the suspension.
    3. Spin the tubes at 10,000 x g at 4 °C for 5 min to discard unlysed tissue and collect supernatants in new tubes. The volume of samples obtained is about 300 μl.
    4. If the tumors are mice xenografts, it is convenient to clarify the samples with Dynabeads protein G in order to eliminate putative mouse Igs that might be infiltrating the tumor thus interfering with phospho-KRAS detection:
      1. Transfer 30 μl of Dynabeads (previously vortexed) to a tube.
      2. Separate the Dynabeads from the solution with Magnet DynaMagTM, discard the supernatant, remove the tube from the magnet and wash dynabeads with λ phosphatase lysis buffer (1 ml, inhibitors free).
      3. Separate the Dynabeads from the buffer with a magnet, discard the buffer, and remove the tube from the magnet.
      4. Incubate the Dynabeads with the samples for 20 min in a tube rotator to allow gentle mixing at 4 °C. The volume of samples is about 300 μl (volume of supernatant obtained in step 3).
      5. Separate the Dynabeads from the samples with the magnet and transfer the clarified samples A and B to a new tube. Measure the volume of clarified samples.
    5. Add 5 mM DTT (from stock 1 M DTT) and 2 mM MnCl2 (from stock 25 mM MnCl2) to clarified samples A and B. Mix gently.
    6. Take 40 μl of sample B to a new tube, add 4 μl of λ Protein phosphatase and incubate at 30 °C for 30 min. The rest of sample B is discarded.
    7. Add phosphatase inhibitors to sample B: 0.2 μl of 1 M sodium fluoride and 0.4 μl of 20 mM sodium orthovanadate (or 0.04 μl of 200 mM sodium orthovanadate) should be added to the 40 μl of sample B to achieve the 5 mM sodium fluoride and 0.2 mM sodium orthovanadate final concentrations that are stated in the Materials section.
    8. Analyse protein content of sample A from step 5 and sample B from step A6 by the Lowry method.

  2. Sample preparation and Phos-tag SDS and transfer
    1. Transfer 20 μg of each sample in 10 μl of final volume with λ phosphatase lysis buffer and add 5 μl of sample buffer 3x. Boil the samples for 1 min in a 1.5 ml tube.
    2. Load the samples into a 12% SDS-PAGE mini gel supplementing the resolving gel with 100 μM Phos-tag and 100 μM MnCl2.
    3. Run the gel 12 h to 15 h at 5 mA/gel at 4 °C.
    4. Soak the gel twice with general transfer buffer containing 1 mM EDTA for 20 min and once with EDTA-free general transfer buffer for 10 min.
    5. Transfer proteins from the gel onto PVDF for 3 h at 70 V.
    6. Perform Western blot with the appropriate antibodies: anti- K-RAS antibodies or any antibody against a putative tag-KRAS expressed.

Representative data

A representative example of Phos-tag SDS-PAGE followed by Western blot using anti-KRAS antibodies, of samples from tumor development in mice after subcutaneously injecting NIH3T3 cells expressing phosphorylatable oncogenic KRAS (KRASG12V S181) is shown in Figure 2 below.


Figure 2. Representative data. 1. K-RasG12V S181 tumor. Non incubated sample; 2. K-RasG12V S181 tumor. Non incubated sample; 3. K-RasG12V S181 tumor. Incubated at 4 °C without phosphatase λ; 4. K-RasG12V S181 tumor. Incubated at 30 °C without phosphatase λ; 5. K-RasG12V S181 tumor. Incubated at 30 °C with phosphatase λ.

Notes

  1. It is important to start from samples with high protein concentration (at least 3 μg/μl) in order to obtain clear results.
  2. When loading the gel avoid using protein molecular weight markers because mobility is not proportional to molecular weight in the presence of Phos-tag reagent.
  3. When loading the gel, leave first and last lanes sample-free to avoid gel distortion.
  4. KRAS phosphorylated band is always retarded compared with unphosphorylated band, but the distance between bands may vary depending on the sample (endogenous KRAS, tag-KRAS expressed in cells or in tumors, etc.). In order to explain correctly the results a control sample treated with λ Phosphatase must be assayed in each experiment at the same time.
  5. Phostag-PAGE gels are quite brittle compared to the normal PAGE gels. Be extra careful when manipulating them.
  6. Load freshly-made samples whenever possible avoiding long-term reloading of the sample. Phospho-KRAS-Phostag complex seems to be sensitive to long-term storage as P-KRAS band would eventually disappear after some time.
  7. Both gel-running conditions and Phos-tag concentration may need to be optimized for each cell line, tumor type, treatment, etc.
  8. It is advisable to run the samples in a usual SDS-PAGE gel (no Phos-tag added) in the same electrophoresis chamber concurrently with the Phos-tag gel since the dye front is difficult to see and follow in the Phos-tag gels.

Recipes

  1. λ phosphatase lysis buffer
    50 mM Tris-HCl (pH 8)
    150 mM NaCl
    2 mM EDTA
    10% glycerol
    1% IGEPAL CA-630
    dH2O
    Add fresh:
    5 mM DTT from stock 1 M DTT
    2 mM MnCl2 from stock 25 mM MnCl2
    Protease and phosphatase inhibitors (for stocks See Materials and Reagents)
    To prepare 5 ml λ phosphatase lysis buffer containing protease and phosphatase inhibitors:
    5 μl aprotinin
    50 μl leupeptin
    50 μl PMSF
    5 μl sodium orthovanadate
    25 μl sodium fluoride
  2. 3x SDS sample buffer
    0.5 M Tris-HCl (pH 6.8)
    1.5 mg Bromophenol blue
    0.60 g SDS
    3 ml glycerol
    3.9 ml mercaptoethanol
    Add dH2O to 10 ml
  3. Electrophoresis running buffer
    25 mM Tris-HCl (pH 8.3)
    192 mM Glycine
    0.1% SDS
  4. Transfer buffer
    25 mM Tris-HCl (pH 8.3)
    192 mM glycine
    0.02 % SDS
    20 % ethanol
  5. 5 mM Phos-tag stock solution
    Resuspend the oily product, Phos-tagTM acrylamide (10 mg) placed in a small plastic tube in 0.10 ml methanol.
    Dilute the solution with 3.2 ml distilled water by pipetting
    Store the solution in the 2-ml microtubes at 4 °C in the dark
  6. 20x PBS (pH 7.2)
    170 g NaCl
    21.5 g Na2HPO4·2H2O
    7.0 g NaH2PO4·H2O
    Add dH2O to 1,000 ml
    For PBS (1x)
    250 ml PBS (20x)
    4,750 ml dH2O
  7. Solution 1 for SDS-PAGE
    Tris-HCl 0.75 M (pH 8.8)
    SDS 0.2 %
  8. Solution 2 for SDS-PAGE
    30% acrylamide
    0.8 % bis acrylamide
  9. Solution 3 for SDS-PAGE
    Tris-HCl 0.25 M (pH 6.8)
    SDS 0.2%
  10. Resolving composition of a 12% SDS-PAGE mini gel (1.5 mm thickness) for Phos-tag SDS
    5 ml solution 1
    4 ml solution 2
    1 ml dH2O
    200 μl Phos-tag stock solution 5 mM
    100 μl MnCl2 stock solution 10 mM
    14 μl Temed
    50 μl APS 13%
  11. Stacking composition of a 12% SDS-PAGE mini gel (1.5 mm thickness)
    0.36 ml solution 2
    1.5 ml solution 3
    1.2 ml dH2O
    7.5 μl Temed
    30 μl APS 13%

Acknowledgments

We are grateful to José Lozano, Associate Professor at Universidad de Málaga (Spain) and to Maribel Geli, Senior Research Scientist at the Molecular Biology Institute of Barcelona (ibmb)-CSIC for providing useful comments during the setting up of the conditions. This technique is part of a study supported by MICINN-Spain (SAF2010-20712) and RTICC (MINECO-Spain; groups RD12/0036/0049, RD12/0036/0031, and RD12/ 0036/0034). C. Barceló and N. Paco are recipients of predoctoral fellowships from MEC-Spain and Generalitat de Catalunya, respectively.

References

  1. Barceló, C., Etchin, J., Mansour, M. R., Sanda, T., Ginesta, M. M., Sanchez-Arevalo Lobo, V. J., Real, F. X., Capella, G., Estanyol, J. M., Jaumot, M., Look, A. T. and Agell, N. (2014). Ribonucleoprotein HNRNPA2B1 interacts with and regulates oncogenic KRAS in pancreatic ductal adenocarcinoma cells. Gastroenterology 147(4): 882-892 e888.
  2. Barceló, C., Paco, N., Morell, M., Alvarez-Moya, B., Bota-Rabassedas, N., Jaumot, M., Vilardell, F., Capella, G. and Agell, N. (2014). Phosphorylation at Ser-181 of oncogenic KRAS is required for tumor growth. Cancer Res 74(4): 1190-1199.

简介

KRAS是在人实体瘤特别是在胰腺,结肠,小肠,胆道和肺中最常突变的癌基因。 我们最近证明,致癌KRAS需要S181磷酸化充分显示其致癌特征,表明其作为对KRAS驱动的肿瘤的治疗性治疗的抑制作用。 由于在人类肿瘤中检测KRAS磷酸化的重要性和不存在针对磷酸化KRAS的特异性抗体,我们开发了基于Phos-tag SDS方法的新方案以检测这种KRAS的翻译后修饰。 Phos标签是特异性结合磷酸化蛋白质的分子,降低其在SDS-PAGE中的迁移速度并允许其从非磷酸化形式分离。

材料和试剂

  1. 人类肿瘤细胞系
  2. 在来自活细胞或组织起源的免疫缺陷小鼠中生长的人肿瘤样品
  3. Phos-tag TM TM丙烯酰胺(Wako Chemicals GmbH,目录号:304-93521,#AAL-107)
  4. λ蛋白磷酸酶(≥400,000单位/ml)(Calbiochem,目录号:539514-20KV)
  5. 抗KRAS(克隆Ab-1)小鼠mAb(Calbiochem,目录号:OP24)或针对假定标签的任何抗体-KRAS表达(HA标签已成功测试)
  6. 蛋白酶抑制剂终浓度
    1. 抑肽酶:150nM(1μg/ml)(Sigma-Aldrich,目录号:A1153)(推荐储备液1mg/ml)
    2. 亮肽素:20μM(10μg/ml)(Sigma-Aldrich,目录号:L2884)(推荐储备液1mg/ml)
    3. 苯基甲磺酰氟(PMSF):1mM(Sigma-Aldrich,目录号:S6508)(推荐储备溶液100mM)
  7. 磷酸酶抑制剂
    1. 原钒酸钠:0.2mM(Sigma-Aldrich,目录号:S6508)(推荐储备液200mM)
    2. 氟化钠:5mM(Sigma-Aldrich,目录号:S7920)(推荐储备液1M)
  8. IGEPAL CA-630(Sigma-Aldrich,目录号:I8896)
  9. 用于免疫沉淀的Dynabeads Protein G(Novex by Life Technologies,目录号:10003D)
  10. Temed
  11. 13%过硫酸铵(APS)
  12. MnCl 2缓冲液10mM和25mM
  13. λ磷酸酶裂解缓冲液(参见配方)
  14. 3x SDS样品缓冲液(见配方)
  15. 电泳缓冲液(见配方)
  16. 传输缓冲区(请参阅配方)
  17. 5 mM Phos标签储备液(见配方)
  18. 20x PBS(请参阅配方)
  19. SDS-PAGE的解决方案1(参见配方)
  20. SDS-PAGE的解决方案2(参见配方)
  21. SDS-PAGE的解决方案3(参见配方)
  22. 解析Phos-tag的12%SDS-PAGE微型凝胶(1.5 mm厚)的组成(参见配方)
  23. 12%SDS-PAGE微型凝胶(1.5mm厚)的堆叠组合物(参见配方)

设备

  1. 组织研磨机(Dounce)(1ml)(Wheaton,目录号:357538)
  2. Magnet DynaMag TM -2(Life Technologies,目录号:12321D)
  3. 管旋转器(Bibby Scientific Ltd.,Stuart SB2)

程序

  1. 样品裂解和λ磷酸酶处理

    所有样品
    1. 对于分析的每个样品(样品A),阴性对照(样品B) 用λ磷酸酶处理。 准备两个 管用冰冷的λ磷酸酶裂解缓冲液,新鲜制备, 含有蛋白酶和磷酸酶抑制剂(A)或仅蛋白酶 抑制剂(B)。


      图1 样品裂解和λ磷酸酶处理的流程图

    对于单元格行
    1. 用10毫升冰冷的磷酸盐缓冲盐水(PBS)从10厘米培养皿(80%汇合)洗涤细胞两次。
    2. 刮细胞与1毫升冰冷PBS,收集在两个1.5毫升microfuge   管A和B(各500μl),并在4℃以1,000xg转速旋转3分钟。 弃去上清液。
    3. 加入18μl冰冷的λ磷酸酶裂解 缓冲液仅含有蛋白酶抑制剂至沉淀B和接下来2μl   的λ蛋白磷酸酶(40单位/μl终浓度)。 孵化 在干燥块中在30℃下干燥30分钟
    4. 加入180μl冰冷的λ 磷酸酶裂解缓冲液含有蛋白酶和磷酸酶抑制剂 到沉淀A.通过吸移和10分钟后重悬浮沉淀 冰,在4℃下以10,000×g离心10分钟。 转移上清液到   新的1.5ml管(样品裂解物A)
    5. 加入160μl冷的λ磷酸酶   裂解缓冲液中含有蛋白酶和磷酸酶抑制剂 颗粒B.重悬沉淀,在冰上10分钟后,旋转10分钟 在4℃下以10,000×g离心。将上清液转移到新的1.5ml管中 (样品裂解物B)
    6. 通过Lowry方法分析从步骤4-5获得的上清液的蛋白质含量。

    对于肿瘤样本
    1. 取相同肿瘤的两个片段,每个约0.1g。 离开 每个肿瘤片段在冰上在0.4ml的存在下在35mm培养皿上 的含有蛋白酶和磷酸酶的冷的λ磷酸酶裂解缓冲液 抑制剂(盘样品A)或仅蛋白酶抑制剂(盘样品B), 但在任何情况下都不是DTT和MnCl 2(将在步骤5中添加)。 剁碎 肿瘤在最小的可能的部分用解剖刀和剪刀和 收集所有体积,包含切碎的组织和裂解缓冲液,在1.5毫升   微量离心管。
      注意:如果样品是冻结的肿瘤, 肿瘤在存在缓冲液时必须进行,而肿瘤是 解冻。
    2. 在冰上用组织研磨机匀化样品,直到悬浮液中没有残留块。
    3. 在4℃下在10,000×g下旋转管5分钟以丢弃未溶解的 组织并收集新管中的上清液。 样品体积 获得约300μl
    4. 如果肿瘤是小鼠异种移植物,它 方便用Dynabeads蛋白G按顺序澄清样品 以消除可能浸润肿瘤的推定的小鼠Ig 从而干扰磷酸化KRAS检测:
      1. 转移30微升Dynabeads(以前涡旋)到管
      2. 使用Magnet DynaMag TM 分离Dynabeads与溶液, 丢弃上清液,从磁铁中取出试管并洗涤 dynabeads用λ磷酸酶裂解缓冲液(1ml,无抑制剂)
      3. 用磁铁将Dynabeads从缓冲液中分离,丢弃缓冲液,并从磁铁上取下管子。
      4. 孵育Dynabeads与样品在管旋转器中20分钟   以允许在4℃温和混合。 样品的体积为约300μl (步骤3中得到的上清液的体积)
      5. 分开 Dynabeads从样品与磁铁和转移澄清 样品A和B到新管。 测量澄清样品的体积。
    5. 向澄清的样品A和B中加入5mM DTT(来自储备1M DTT)和2mM MnCl 2(来自储备25mM MnCl 2)。轻轻混合。 />
    6. 取40μl的样品B到新管,加入4μl的λ蛋白 磷酸酶,并在30℃孵育30分钟。 样品B的其余部分 舍弃。
    7. 向样品B中加入磷酸酶抑制剂:0.2μl的1M 氟化钠和0.4μl20mM原钒酸钠(或0.04μl 200 mM原钒酸钠)加入到40μl样品B中   达到5mM氟化钠和0.2mM原钒酸钠最终 浓度,在材料部分中说明。
    8. 通过Lowry方法分析来自步骤5的样品A和来自步骤A6的样品B的蛋白质含量。

  2. 样品制备和Phos-tag SDS和转移
    1. 转移20微克每个样品在10微升的最终体积与λ 磷酸酶裂解缓冲液,加入5μl样品缓冲液3x。 煮沸 样品在1.5ml管中1分钟
    2. 将样品加载到用100μMPhos标签和100μMMnCl 2补充分辨凝胶的12%SDS-PAGE微型凝胶中。
    3. 在4℃下以5mA /凝胶运行凝胶12小时至15小时
    4. 用含有1mM EDTA的普通转移缓冲液浸泡凝胶两次   20分钟,用无EDTA一般转移缓冲液洗涤10分钟
    5. 将蛋白质从凝胶转移到PVDF在70 V下3小时
    6. 用适当的抗体:抗K-RAS进行Western印迹 抗体或针对推定的标签KRAS表达的任何抗体。

代表数据

在皮下注射表达可磷酸化的致癌KRAS(KRASG12V S181)的NIH3T3细胞后,来自小鼠中肿瘤发展的样品的Phos标签SDS-PAGE,随后使用抗KRAS抗体的蛋白质印迹的代表性实例显示于图2中。 >

图2。 代表数据 。 1. K-RasG12V S181肿瘤。非温育样品; 2.K-RasG12V S181肿瘤。非温育样品; 3.K-RasG12V S181肿瘤。在4℃孵育,无磷酸酶λ; 4.K-RasG12V S181肿瘤。在30℃孵育,无磷酸酶λ; 5. K-RasG12V S181肿瘤。在30℃下用磷酸酶λ孵育。

笔记

  1. 从具有高蛋白质浓度的样品(至少3μg/μl)开始是重要的,以获得清楚的结果
  2. 当加载凝胶时避免使用蛋白质分子量标记,因为在存在Phos-标签试剂的情况下,迁移率与分子量不成比例。
  3. 加载凝胶时,离开第一和最后一道样品,避免凝胶变形
  4. KRAS磷酸化带与未磷酸化带相比总是被延迟,但是带之间的距离可以根据样品(内源性KRAS,在细胞中或在肿瘤中表达的标签-KRAS等)而变化。为了正确解释结果,用λ磷酸酶处理的对照样品必须在每个实验中同时进行测定。
  5. Phostag-PAGE凝胶与正常PAGE凝胶相比相当脆弱。在操作它们时要格外小心。
  6. 尽可能地装载新制的样品,避免样品长期重新装载。磷酸-RKAS-Phostag复合物似乎对长期储存敏感,因为P-KRAS带最终在一段时间后消失。
  7. 凝胶运行条件和Phos标签浓度可能需要对每种细胞系,肿瘤类型,治疗等进行优化。
  8. 建议在与Phos标记凝胶同时在同一电泳室中在通常的SDS-PAGE凝胶(不加入Phos-标签)中运行样品,因为染料前沿在Phos标记中难以看到和跟踪 凝胶。

食谱

  1. λ磷酸酶裂解缓冲液
    50mM Tris-HCl(pH8)
    150mM NaCl 2mM EDTA 10%甘油 1%IGEPAL CA-630
    dH 2 2 O 新添加:
    来自库存1 M DTT的5mM DTT
    2mM MnCl 2来自原料25mM MnCl 2/v/v 蛋白酶和磷酸酶抑制剂(股票参见材料和试剂)
    制备含蛋白酶和磷酸酶抑制剂的5ml磷酸酶裂解缓冲液:
    5μl抑肽酶
    50μl亮抑酶肽
    50μlPMSF
    5μl原钒酸钠
    25μl氟化钠
  2. 3x SDS样品缓冲液
    0.5M Tris-HCl(pH 6.8)
    1.5mg溴酚蓝
    0.60克SDS
    3ml甘油 3.9毫升巯基乙醇 将dH 2加到10ml ml/h
  3. 电泳运行缓冲液
    25mM Tris-HCl(pH8.3)
    192mM甘氨酸 0.1%SDS
  4. 传输缓冲区
    25mM Tris-HCl(pH8.3)
    192 mM甘氨酸 0.02%SDS
    20%乙醇
  5. 5 mM Phos标签储液
    将置于0.10ml甲醇中的小塑料管中的油状产物Phos-tag TM TM丙烯酰胺(10mg)重悬。
    通过移液
    用3.2ml蒸馏水稀释溶液 将溶液储存在2 ml微管中,在4°C在黑暗
  6. 20x PBS(pH 7.2)
    170克NaCl
    21.5g Na 2 HPO 4 H 2 2H 2 O
    7.0g NaH 2 PO 4 PO 4·H 2 O
    将dH <2> O添加至1,000 ml
    对于PBS(1x)
    250 ml PBS(20x)
    4,750ml dH 2 O n /
  7. SDS-PAGE的解决方案1
    Tris-HCl 0.75M(pH 8.8)
    SDS 0.2%
  8. SDS-PAGE的解决方案2
    30%丙烯酰胺 0.8%双丙烯酰胺
  9. SDS-PAGE的解决方案3
    Tris-HCl 0.25M(pH 6.8)
    SDS 0.2%
  10. 用于Phos-tag SDS的12%SDS-PAGE微凝胶(1.5mm厚)的组成 5ml溶液1
    4 ml溶液2
    1ml dH 2 O 2 / 200μlPhos标签储备液5 mM
    100μlMnCl 2储备溶液10mM
    14μl温度
    50微升APS 13%
  11. 12%SDS-PAGE微凝胶(1.5mm厚)的堆叠组合物 0.36ml溶液2
    1.5ml溶液3
    1.2ml dH 2 O 2 / 7.5μl温度
    30微升APS 13%

致谢

我们感谢马拉加大学(西班牙)副教授JoséLozano和巴塞罗那分子生物学研究所(ibmb)高级研究科学家Maribel Geli在设立条件时提供有用的意见。该技术是由MICINN-Spain(SAF2010-20712)和RTICC(MINECO-Spain;组RD12/0036/0049,RD12/0036/0031和RD12/0036/0034)支持的研究的一部分。 C.Barceló和N. Paco分别是来自MEC-Spain和Generalitat de Catalunya的preoctoral奖学金的获得者。

参考文献

  1. Barceló,C.,Etchin,J.,Mansour,MR,Sanda,T.,Ginesta,MM,Sanchez- Arevalo Lobo,VJ,Real,FX,Capella,G.,Estanyol,JM,Jaumot, AT和Agell,N。(2014)。 核糖核蛋白HNRNPA2B1与胰腺导管腺癌细胞中的致癌性KRAS相互作用并调节致癌性KRAS。 Gastroenterology 147(4):882-892 e888。
  2. Barceló,C.,Paco,N.,Morell,M.,Alvarez-Moya,B.,Bota-Rabassedas,N.,Jaumot,M.,Vilardell,F.,Capella,G。和Agell, )。 肿瘤生长需要致癌KRAS的Ser-181处的磷酸化。 Cancer Res 74(4):1190-1199。
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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。
Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Barceló, C., Paco, N., Cabot, D., Garrido, E., Agell, N. and Jaumot, M. (2015). Detection of Phospho-KRAS by Electrophoretic Mobility Change in Human Cell Lines and in Tumor Samples from Nude Mice Grafts. Bio-protocol 5(6): e1421. DOI: 10.21769/BioProtoc.1421.
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