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Immunoprecipitation of ROR1
ROR1的免疫共沉淀

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Abstract

ROR1 is a receptor tyrosine kinase family member studied for its roles in development and cancer. Here we describe a protocol for immunoprecipitation of endogenous ROR1 from t(1;19) (a disease subtype categorized by its chromosome translocation) acute lymphoblastic leukemia immortalized cell lines.

Materials and Reagents

  1. ROR1-positive cells (e.g. RCH-ACV cells, Kasumi-2 cells, 697 cells)
  2. Cell lysis buffer (Cell Signaling Technology, catalog number: 9803 )
  3. Protease inhibitor cocktail (cOmplete Mini EDTA-free) (Roche, catalog number: 04693159001 )
  4. Phosphatase inhibitor cocktail II (Sigma-Aldrich, catalog number: P5726 )
  5. Antibody specific for ROR1 (R&D Systems, catalog number: AF2000 )
  6. Isotype matched control (R&D Systems, catalog number: AB-108-C )
  7. Protein G Agarose beads (EMD Millipore, catalog number: 16-266 )
  8. SDS
  9. BSA
  10. DTT

Equipment

  1. P1000 pipette
  2. Centrifuge

Procedure

  1. 107 cells (such as RCH-ACV, Kasumi-2, 697 cells) are pelleted, washed 1x in PBS, pelleted, and thoroughly resuspended in 500 μl ice-cold lyses buffer supplemented with protease inhibitor cocktail and phosphatase inhibitor cocktail II (according to manufacturers’ instruction) using a P1000 pipette.
  2. The lysis reaction is kept on ice and vortexed at max speed 3 times for 3-second bursts. The reaction is kept on ice for 5 min between each vortexing.
  3. Following lysis, samples are centrifuged at max-speed for 15 min at 4 °C.
  4. Following pelleting, the supernatant is transferred to a fresh tube, kept on ice, and subjected to Bradford protein concentration analysis. Lysate is diluted to 2 mg/ml in lysis buffer for IP.
  5. 3 μg of antibody specific for ROR1 or an isotype matched control was incubated with cell extracts (500 μl at 2 mg/ml) by rotating overnight.
  6. Protein G Agarose beads were prepared for precipitation by washing twice in lysis buffer and blocking for 1 h (nutating) at 4 °C in lysis buffer containing 0.5% BSA.
  7. Following blocking, beads were pelleted and resuspended in lysis buffer to a 50% slurry, and 40 μl 50% slurry was added to lysate and rocked at 4 °C for 1 h.
  8. Following precipitation, beads were washed in 500 μl lysis buffer 3x by nutating at 4 °C for 20 min.
  9. Proteins were eluted from the beads by boiling in an aqueous solution with 2% SDS and 25 mM DTT.

Acknowledgments

This protocol was adapted from Bicocca et al. (2012).

References

  1. Bicocca, V. T., Chang, B. H., Masouleh, B. K., Muschen, M., Loriaux, M. M., Druker, B. J. and Tyner, J. W. (2012). Crosstalk between ROR1 and the Pre-B cell receptor promotes survival of t(1;19) acute lymphoblastic leukemia. Cancer Cell 22(5): 656-667.

简介

ROR1是受体酪氨酸激酶家族成员,研究其在发展和癌症中的作用。 在这里我们描述了内源性ROR1从t(1; 19)(由其染色体易位分类的疾病亚型)急性淋巴细胞白血病永生化细胞系免疫沉淀的协议。

材料和试剂

  1. ROR1阳性细胞(例如,RCH-ACV细胞,Kasumi-2细胞,697个细胞)
  2. 细胞裂解缓冲液(Cell Signaling Technology,目录号:9803)
  3. 蛋白酶抑制剂混合物(cOmplete Mini EDTA-free)(Roche,目录号:04693159001)
  4. 磷酸酶抑制剂混合物II(Sigma-Aldrich,目录号:P5726)
  5. ROR1特异性抗体(R& D Systems,目录号:AF2000)
  6. 同种型匹配对照(R& D Systems,目录号:AB-108-C)
  7. 蛋白G琼脂糖珠(EMD Millipore,目录号:16-266)
  8. SDS
  9. BSA
  10. DTT

设备

  1. P1000移液器
  2. 离心机

程序

  1. 沉淀10×10 7个细胞(例如RCH-ACV,Kasumi-2,697细胞),在PBS中洗涤1次,沉淀,并彻底重悬于补充有蛋白酶抑制剂混合物的500μl冰冷的裂解缓冲液 和磷酸酶抑制剂混合物II(根据制造商的说明书)使用P1000移液管。
  2. 裂解反应保持在冰上,并以最大速度3次涡旋3秒钟。 在每次涡旋之间将反应在冰上保持5分钟。
  3. 裂解后,将样品在4℃下以最大速度离心15分钟。
  4. 在造粒后,将上清液转移到新鲜试管中,保存在冰上,并进行Bradford蛋白质浓度分析。 将溶胞产物在用于IP的裂解缓冲液中稀释至2mg/ml。
  5. 将3μg特异于ROR1的抗体或同种型匹配的对照通过旋转过夜与细胞提取物(500μl,2mg/ml)孵育。
  6. 蛋白G通过在裂解缓冲液中洗涤两次并在4℃下在含有0.5%BSA的裂解缓冲液中封闭1小时(章动)来制备用于沉淀的琼脂糖珠。
  7. 封闭后,将珠粒化并重悬于裂解缓冲液中至50%浆液,并将40μl50%浆液加入裂解物中并在4℃摇动1小时。
  8. 沉淀后,通过在4℃下章动20分钟在500μl裂解缓冲液3x中洗涤珠子。
  9. 通过在含有2%SDS和25mM DTT的水溶液中煮沸从珠上洗脱蛋白质

致谢

该方案改编自Bicocca等人(2012)。

参考文献

  1. Bicocca,V.T.,Chang,B.H.,Masouleh,B.K.,Muschen,M.,Loriaux,M.M.,Druker,B.J。和Tyner,J.W。(2012)。 ROR1和Pre-B细胞受体之间的串扰促进t(1; 19)急性淋巴细胞存活 白血病。 癌细胞 22(5):656-667
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引用:Bicocca, V. T. and Tyner, J. W. (2013). Immunoprecipitation of ROR1. Bio-protocol 3(18): e904. DOI: 10.21769/BioProtoc.904.
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