参考讲稿cst讲座

1、 eractionGeneticYeast 2-hybrid(母 eractionGeneticYeast 2-hybrid(母 Biochemical Co-ion免疫q沉淀 Cell- FRET - fluorescence resonance energy trasfer振能量 Rosetta Co-Co-用针对一n蛋Co-用针对一n蛋白的抗体去w获P该蛋白可能结合的其它蛋白Determination of Protein-ionofproteinXwithsepharose+Sepharose beads ProteinbstoproteinXisapartofaDeterminati

2、on of Protein-ionofproteinXwithsepharose+Sepharose beads ProteinbstoproteinXisapartofaproteinXisasingleCollectDissolve oCo-pptofY&Zwith SDS ProteinProteinY Protein1D1DCo-Co-磷酸化O点分磷酸化O点分缺y分析P缺y分析Pco-IP联磷酸化O点分磷酸化O点分Step By StepStep By Step Guide to 品的预纯化eP目标蛋白抗体q孵免疫q沉oWestern blot 分Step By Step Guide

3、to SlepreparationStep By Step Guide to SlepreparationandcellPreclearing of the Incubation ntibodyandformationofantigen ion of the complex Washing of the antibody-antigen complexes to non-specific proteins and ysis of complexes/antigen erest by SDS-1.lepreparationand1.lepreparationandcell1.a. Non-den

4、aturing lysis buffer非变性裂解液 RIPA(Radio extracts，提物ionAssay)buffer Denaturing lysis buffer/buffer for non-detergent 变性裂解液 (e.g.Histone，蛋白 1.lepreparation 1.lepreparationandcell 2.temperature3. 4.NaCl150 510glycerol甘油 6.Proteaseinhibitor 7Cation某些特别的离子ATP, 8.BestfreshlyStep By Step Guide to Slepreparat

5、ionStep By Step Guide to SlepreparationandcellPreclearing of the Incubation ntibodyandformationofantigen ion of the complex Washing of the antibody-antigen complexes to non-specific proteins and ysis of complexes/antigen erest by SDS-2.Preclearing of the2.Preclearing of the 减少裂解混合物中的非特异性蛋白的含 同时也减少了可

6、能与ProteinA/ProteinG发生交叉反应的蛋白含量 2.Preclearing of the2.Preclearing of the 同时加入Protein 纯化后一定将o或无s抗体尽量oStep By Step Guide to SlepreparationStep By Step Guide to SlepreparationandcellPreclearing of the Incubation ntibodyandformationofantigen ion of the complex Washing of the antibody-antigen complexes to

7、 non-specific proteins and ysis of complexes/antigen erest by SDS-3. IP: Incubation3. IP: IncubationAntibody and Formation Antibody-Antigen N操作 100-500 g 胞裂解 同时加入适量一抗抗体量浓度要通过预实验或查阅文献和说明书g确定3. IP: Incubation3. IP: IncubationAntibody and Formation Antibody-Antigen O要加太的一抗，否则背景较高Step By Step Guide to S

8、lepreparationStep By Step Guide to SlepreparationandcellPreclearing of the Incubation ntibodyandformationofantigen ion of the complex Washing of the antibody-antigen complexes to non-specific proteins and ysis of complexes/antigen erest by SDS- 4.ionofthecomplex 4.ionofthecomplex ProteinAProteinG的混合

9、物将枪剪r粗口并烧圆，k加 4.ionof 4.ionofthecomplex Step By Step Guide to SlepreparationStep By Step Guide to SlepreparationandcellPreclearing of the Incubation ntibodyandformationofantigen ion of the complex Washing of the antibody-antigen complexes to non-specific proteins and ysis of complexes/antigen erest

10、by SDS-5. Washing of5. Washing of the antibody-antigen 充分洗涤很重O断增加NaCl的浓度和去垢剂的浓度系列洗涤Step By Step Guide to SlepreparationStep By Step Guide to SlepreparationandcellPreclearing of the Incubation ntibodyandformationofantigen ion of the complex Washing of the antibody-antigen complexes to non-specific pr

11、oteins and ysis of complexes/antigen erest by SDS-ysisofcomplexes/antigen erestSDS-ysisofcomplexes/antigen erestSDS-P， 一抗或将IPo一抗P磁珠交联后再洗脱MouseAnti-rabbitIgG(ConformationSpecific)(L27A9)mAb Mouse Anti-rabbit IgG (Light-Chain Specific) (L57A3) mAbMouseAnti-rabbitIgG(ConformationSpecific)(L27A9)mAb(HRP

12、IPtroubleshooting:HighIPtroubleshooting:High制_样品中可能有O制_品后离心纯化，No后后续试验洗涤O次洗涤，并增加洗涤缓液o的NaCl和去垢剂浓度预理以k非特异性吸IPtroubleshooting:HighIPtroubleshooting:High抗体本身特异性O好g件优化减抗使用了过多的胞或进行裂解减胞或量，s们推100-500ug胞裂解物IPtroubleshooting:HighIPtroubleshooting:High减IP时加入的胞裂解物对胞裂解物预IP troubleshooting: IP troubleshooting: High amount antibody 减抗体用量，或将抗体P磁珠交