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On-bead Digestion:从免疫沉淀到质谱分析
作者:Morey 发布时间:2017-09-15 点击次数:2891

  

图一.jpg


    On-bead Digestion:从免疫沉淀到质谱分析

    免疫沉淀蛋白及其相互作用物到质谱分析的转移率应尽可能高。这对于低丰度的蛋白质尤其重要。

    通过使用on-bead-digestion,您能够:

  ·节省时间——你不需要洗提结合蛋白质

  ·保持蛋白质高浓度——不要稀释你的样品

  ·更少的洗涤步骤能更好的保留Co-IP的相互作用物

  

图2.胰蛋白酶.jpg


  胰蛋白酶

  对Nano-Traps (GFP-Trap, RFP-Trap, mNeonGreen-Trap, MBP-Trap, GST-Trap,等)的消化会产生少量的缩氨酸。这是由于固定化的羊驼抗体的小尺寸(14 kDa)导致的。例如,用胰蛋白酶对GFP-Trap进行on-bead-digestion只产生4个缩氨酸。

  这并不是唯一的优势:由于它们的高亲和力和低背景,ChromoTek的Nano-Traps具有非凡的IP/ Co-IP性能。

  ChromoTek的Nano-Traps是分离自羊驼单域抗体(VHHs,也称为纳米体)。因此,它们缺乏抗体轻链,产生更纯粹的IP/ Co-IP结果。

  „The best results were obtained by direct tryptic digest of the material on beads followed by mass spectrometry”, Zoltan Lipinszki et al. (2014)

  ChromoTek GFP-Trap的on-bead-digestion protocol请点击下方查看:

  

图3.下载说明书.jpg

  参考文献:

  1. Arne H. Smits, Pascal W. T. C. Jansen, Ina Poser, Anthony A. Hyman, Michiel Vermeulen; Stoichiometry of chromatin-associated protein complexes revealed by label-free quantitative mass spectrometry-based proteomics. Nucleic Acids Res 2013; 41 (1): e28. doi: 10.1093/nar/gks941

  2. Zoltan Lipinszki, Peng Wang, Rhys Grant, Catherine Lindon, Nikola S. Dzhindzhev, Pier Paolo D’Avino, Marcin R. Przewloka, David M. Glover, Vincent Archambault; Affinity Purification of Protein Complexes from Drosophila Embryos in Cell Cycle Studies. Methods Mol Biol. 2014;1170:571-88. doi: 10.1007/978-1-4939-0888-2_33.

  3. Susan L. Kloet, Matthew M. Makowski, H. Irem Baymaz, Lisa van Voorthuijsen, Ino D. Karemaker, Alexandra Santanach, Pascal W.T.C. Jansen, Luciano Di Croce, Michiel Vermeulen; The dynamic interactome and genomic targets of Polycomb complexes during stem cell differentiation. Nat Struct Mol Biol. 2016 July ; 23(7): 682–690. doi:10.1038/nsmb.3248

  4. Benedetta Turriziani, Amaya Garcia-Munoz, Ruth Pilkington, Cinzia Raso, Walter Kolch, Alexander von Kriegsheim; On-Beads Digestion in Conjunction with Data-Dependent Mass Spectrometry: A Shortcut to Quantitative and Dynamic Interaction Proteomics. Biology 2014, 3(2), 320-332; doi:10.3390/biology3020320


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