參考講稿cst講座

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4、aturing lysis buffer非變性裂解液 RIPA(Radio extracts，提物ionAssay)buffer Denaturing lysis buffer/buffer for non-detergent 變性裂解液 (e.g.Histone，蛋白 1.lepreparation 1.lepreparationandcell 2.temperature3. 4.NaCl150 510glycerol甘油 6.Proteaseinhibitor 7Cation某些特別的離子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 減少裂解混合物中的非特異性蛋白的含 同時也減少了可

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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 同時加入適量一抗抗體量濃度要通過預(yù)實驗或查閱文獻(xiàn)和說明書g確定3. IP: Incubation3. IP: IncubationAntibody and Formation Antibody-Antigen O要加太的一抗，否則背景較高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、物將槍剪r粗口并燒圓，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 充分洗滌很重O斷增加NaCl的濃度和去垢劑的濃度系列洗滌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

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12、IPtroubleshooting:HighIPtroubleshooting:High制_樣品中可能有O制_品后離心純化，No后后續(xù)試驗洗滌O次洗滌，并增加洗滌緩液o的NaCl和去垢劑濃度預(yù)理以k非特異性吸IPtroubleshooting:HighIPtroubleshooting:High抗體本身特異性O(shè)好g件優(yōu)化減抗使用了過多的胞或進(jìn)行裂解減胞或量，s們推100-500ug胞裂解物IPtroubleshooting:HighIPtroubleshooting:High減IP時加入的胞裂解物對胞裂解物預(yù)IP troubleshooting: IP troubleshooting: High amount antibody 減抗體用量，或?qū)⒖贵wP磁珠交