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The IA-2 interactome

The IA-2 interactome. Qine-Zhu. INTRODUCTION. Islet antigen. Islet antigen-2 (IA-2). 1-576. 577-600. 601-979. transmembrane protein of tyrosine phosphatase (PTP) family. Material. Yeast(AH109). HEK 293 CELL. Method. Yeast two-hybrid system. Immunoprecipitation. His, β-gal.

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The IA-2 interactome

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  1. The IA-2 interactome Qine-Zhu

  2. INTRODUCTION

  3. Islet antigen

  4. Islet antigen-2 (IA-2) 1-576 577-600 601-979 transmembrane protein of tyrosine phosphatase (PTP) family

  5. Material Yeast(AH109) HEK 293 CELL

  6. Method Yeast two-hybrid system Immunoprecipitation

  7. His, β-gal

  8. 酵母双杂交基本过程

  9. 免疫共沉淀实验流程 （1） 转染后24-48 h 可收获细胞，加入适量细胞裂解缓冲液（含蛋白酶抑制剂），冰上裂解30min, 细胞裂解液于4°C， 最大转速离心30 min后取上清。 （2） 取少量裂解液以备Western blot分析，剩余裂解液加1μg相应的抗体加入到细胞裂解液，4°C缓慢摇晃孵育过夜。 （3） 取10μl protein A 琼脂糖珠，用适量裂解缓冲液洗3 次，每次3,000 rpm离心3 min。 （4） 将预处理过的10μl protein A 琼脂糖珠加入到和抗体孵育过夜的细胞裂解液中4°C缓慢摇晃孵育2-4h，使抗体与protein A琼脂糖珠偶连。 （5） 免疫沉淀反应后，在4°C 以3,000 rpm 速度离心3 min，将琼脂糖珠离心至管底。将上清小心吸去，琼脂糖珠用1ml裂解缓冲液洗3－4次。最后加入15μl的2×SDS 上样缓冲液，沸水煮5分钟。 （6） SDS-PAGE, Western blotting或质谱仪分析.

  10. RESULTS

  11. Co-immunoprecipitation in mammalian cells of IA-2 and interacting proteins

  12. Conclusion The IA-2 interactome currently consists of 12 interacting proteins Deletion of the Cterminal of IA-2 abolished the interaction between IA-2 and MADD, as did deletion of 60 amino acids from the N-terminal part (aa 804–979) of the best IA-2- interacting mutant (aa 744–979). A similar (but weaker)binding profile to IA-2 was found with SNX19. Only SNX19 containing the PX domain (aa 494–992) bound to IA-2. The binding of PTPρ to IA-2 was stronger than the binding of PTPσ to IA-2, as measured by α-galactosidase activity.

  13. THANK YOU! Your question are appreciated!

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