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 On-bead Digestion:從免疫沉淀到質(zhì)譜分析

 免疫沉淀蛋白及其相互作用物到質(zhì)譜分析的轉(zhuǎn)移率應(yīng)盡可能高。這對于低豐度的蛋白質(zhì)尤其重要。

 通過使用on-bead-digestion,您能夠：

節(jié)省時(shí)間 你不需要洗提結(jié)合蛋白質(zhì)

保持蛋白質(zhì)高濃度 不要稀釋你的樣品

更少的洗滌步驟能更好的保留Co-IP的相互作用物

胰蛋白酶

對Nano-Traps (GFP-Trap, RFP-Trap, mNeonGreen-Trap, MBP-Trap, GST-Trap,等)的消化會(huì)產(chǎn)生少量的縮氨酸。這是由于固定化的羊駝抗體的小尺寸(14 kDa)導(dǎo)致的。例如，用胰蛋白酶對GFP-Trap進(jìn)行on-bead-digestion只產(chǎn)生4個(gè)縮氨酸。

這并不是*的優(yōu)勢:由于它們的高親和力和低背景，ChromoTek的Nano-Traps具有非凡的IP/ Co-IP性能。

ChromoTek的Nano-Traps是分離自羊駝單域抗體(VHHs，也稱為納米體)。因此，它們?nèi)狈贵w輕鏈，產(chǎn)生更純粹的IP/ Co-IP結(jié)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請點(diǎn)擊下方查看：

參考文獻(xiàn)：

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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