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
(*contributed equally to this work) 发布: 2015年03月20日第5卷第6期 DOI: 10.21769/BioProtoc.1421 浏览次数: 9645
评审: HongLok LungThomas J. BartoshShalini Low-Nam
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参见作者原研究论文
The authors used this protocol in:
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
- Human tumor cell lines
- Human tumor samples grown in immunodeficient mice from living cells or tissue origins
- Phos-tagTM acrylamide (Wako Chemicals GmbH, catalog number: 304-93521 , #AAL-107)
- λ Protein phosphatase (≥400,000 units/ml) (Calbiochem, catalog number: 539514-20KV )
- 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)
- Protease inhibitors final concentrations
- Aprotinin: 150 nM (1 μg/ml) (Sigma-Aldrich, catalog number: A1153 ) (recommended stock solution 1 mg/ml)
- Leupeptin: 20 μM (10 μg/ml) (Sigma-Aldrich, catalog number: L2884 ) (recommended stock solution 1 mg/ml)
- Phenylmethanesulfonyl fluoride (PMSF): 1 mM (Sigma-Aldrich, catalog number: S6508 ) (recommended stock solution 100 mM)
- Aprotinin: 150 nM (1 μg/ml) (Sigma-Aldrich, catalog number: A1153 ) (recommended stock solution 1 mg/ml)
- Phosphatase inhibitors
- Sodium orthovanadate: 0.2 mM (Sigma-Aldrich, catalog number: S6508) (recommended stock solution 200 mM)
- Sodium fluoride: 5 mM (Sigma-Aldrich, catalog number: S7920 ) (recommended stock solution 1 M)
- Sodium orthovanadate: 0.2 mM (Sigma-Aldrich, catalog number: S6508) (recommended stock solution 200 mM)
- IGEPAL CA-630 (Sigma-Aldrich, catalog number: I8896 )
- Dynabeads Protein G for immunoprecipitation (Novex by Life Technologies, catalog number: 10003D )
- Temed
- 13% ammonium persulfate (APS)
- MnCl2 stock solutions 10 mM and 25 mM
- λ phosphatase lysis buffer (see Recipes)
- 3x SDS sample buffer (see Recipes)
- Electrophoresis running buffer (see Recipes)
- Transfer buffer (see Recipes)
- 5 mM Phos-tag stock solution (see Recipes)
- 20x PBS (see Recipes)
- Solution 1 for SDS-PAGE (see Recipes)
- Solution 2 for SDS-PAGE (see Recipes)
- Solution 3 for SDS-PAGE (see Recipes)
- Resolving composition of a 12% SDS-PAGE mini gel (1.5 mm thickness) for Phos-tag (see Recipes)
- Stacking composition of a 12% SDS-PAGE mini gel (1.5 mm thickness) (see Recipes)
Equipment
- Tissue grinder (Dounce) (1 ml) (Wheaton, catalog number: 357538 )
- Magnet DynaMagTM-2 (Life Technologies, catalog number: 12321D )
- Tube Rotator (Bibby Scientific Ltd., Stuart SB2)
Procedure
文章信息
版权信息
© 2015 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
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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分类
癌症生物学 > 通用技术 > 生物化学试验
分子生物学 > 蛋白质 > 检测
生物化学 > 蛋白质 > 活性
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