CO-IP实验流程 1、细胞裂解液 2、加入偶联抗体孵育， 3、抗原及其相互作用 或蛋白质混合物 并过柱子 蛋白吸附于柱子 Schematic of a standard co-imaunoprecipitat (co-IP) 4、洗脱，获得抗原 Coupled Antibody 及其相互作用蛋白 Antigen Protein Interacting with Antigen 5、SDS-PAGE/WB/质 谱分析
CO-IP实验流程 1、细胞裂解液 或蛋白质混合物 2、加入偶联抗体孵育， 并过柱子 3、抗原及其相互作用 蛋白吸附于柱子 4、洗脱，获得抗原 及其相互作用蛋白 5、SDS-PAGE/WB/质 谱分析
CO-IP应用示例 Smad3与TRB3相互作用 p62与TRB3相互作用 a b TRB3-HA + TRB3-HA Normal rabbit IgG Anti-p62 Smad3-myc p62-DDK + IP: a-HA 40 HA Γ40 TRB3 IP:a-myc IP:DDK L55 DDK 55 p62 a-myc HA -lgG HC 40 a-HA Input 40 TRB3 Cell Lysate L55 DDK indul a-myc 55 p62 Research Article 3235 ARTICLE TRB3 interacts with SMAD3 promoting tumor cell Recelved 16 May 2015 Accepted 29 n 2015 Published Aug 201 D 10.1038/ncomms9s OPEN migration and invasion TRB3 links insulin/IGF to tumour promotion by Wngnong Yng.n Chen interacting with p62 and impeding autophagic/ proteasomal degradations Fang HuaKe Jiao-Jiao Yu,Xiao-Xi Lv,Jun Yan,Xiao-Wei Zhang,Wei Sun'Heng Lin',Shuang Shang Feng Wang'.Bing Cui.Rong Mu,Bo Huang Jian-Dong Jiang!2&Zhuo-Wei Hu!
CO-IP应用示例 Smad3与TRB3相互作用 p62与TRB3相互作用
染色质免疫共沉淀 (Chromatin immunoprecitation ChIP) 2 Crosslink Protein Cell Nucleus Add Protein- and Shear DNA 3 Specific Antibody 罐W ACTGGTGACAGGACG Immunoprecipitate and purify complexes 6 Sequence DNA fragment and map to genome 5 Reverse Crosslinks Purify DNA and prepare for sequencing
染色质免疫共沉淀 (Chromatin immunoprecitation，ChIP)
ChIP实验流程示意图 Cross-link protein to DNA in Break open cells and shear DNA by Add primary living cells with formaldehyde Sonication or Enzymatic Shearing antibody of interest Transfer DNA into fresh tubes: Reverse protein/DNA Capture antibody-bound DNA is ready for analysis cross-links,treat with protein/DNA complexes and Proteinase K wash using Magnetic Beads Schematic of chromatin immunoprecipitation(ChlP)using ChIP-IT Express. In ChlP,protein/DNA interactions are fixed,and the DNA is sheared and precipitated using an antibody and Protein G Magnetic Beads.Following cross-link reversal,the DNA is screened to determine which gene(s)were bound by the protein of interest
ChIP实验流程示意图