细胞信号转导的技术方法

细胞信号转导的技术方法蛋白激酶磷酸化及其活性测定蛋白激酶磷酸化的检测裂解培养的细胞获得裂解上清以免疫共沉淀法获得蛋白激酶SDS再加入蛋白激酶磷酸化特异性抗体以Phospho

Ab-HRPWestern化学发光检测试剂盒测定蛋白激酶的磷酸化程度。经典蛋白激酶活性分析实验流程以免疫共沉淀法获得蛋白激酶加入该激酶底物和32-ATP，激酶使底物磷酸化放射自显影，确定磷酸化条带以免疫共沉淀法获得蛋白激酶加入该激酶底物，激酶使底物磷酸化再加入底物磷酸化特异性抗体以Phospho

Ab-HRPWestern化学发光检测试剂盒测定底物的磷酸化程度，进而推出蛋白激酶活性非放射性蛋白激酶活性分析实验流程较传统激酶活性测定方法的优点无需接触放射性物质敏感度高：可以检测到每个磷酸化分子特异性：利用磷酸化特异性抗体可以进行位点特异性分析信噪比高低背景系统完整：提供一整套直接从细胞裂解液中测试酶活的试剂节约时间：由几天降到几分钟02468101214051020306090120Time(min)RelativekinaseactivityDynamicprocessesofp38activationbyLPSinRAWcellsEffectofindividualMAPKpathwaysonPRAKactivityinintactcellsMKK7(D)GST-ATF2GST-ELK1MKK1(E)MKK6(E)ControlHSP27PRAKControlAniso.ArseniteTNF-80kDa-47kDa-39kDa-80kDa-47kDakDa97.4-68.0-43.9-29.0-18.4-14.3-ControlAniso.ArseniteTNFABThemajorkinasesofp38andp38P38MAPKinasePathway蛋白磷酸化位点分析及其功能鉴定PhosphopeptidemapofHSP27phosphorylatedbyPRAKinvitro

ChromatographyElectrophoresispH1.9+MAPKAPK2ChromatographyElectrophoresispH1.9+PRAKChromatographyElectrophoresispH1.9MIX+++++----+-HSP27p38GST-PRAK(93A)GST-PRAK(WT)GST-PRAK(186A)GST-PRAK(212A214A)GST-PRAK(182A)GST-PRAK(WT)GST-PRAK(182D)GST-PRAK(182D212D)+-+-+-FoldofActivation05101520GST-PRAK(182A)GST-PRAK(182D)GST-PRAK(WT)GST-PRAK(93A)GST-PRAK(186A)GST-PRAK(182D212D)GST-PRAK(212A214A)BAElectrophoresispH8.9++p38-PRAK(182A)+p38-PRAK(182D)p38-PRAK(wt)T182istheregulatoryphosphorylationsiteofPRAKDNA重组技术的应用活性诱变体无活性诱变体结构与功能的研究PCRprimerPCRprimerJNK2JNK3JNK1p38p38p38p38ERK1ERK2ERK5TherelationshipbetweenthemembersofMAPKfamilyMAPK

Dural

PhosphorylationSitesL-12Length

**hp38

DFGLARHTDDEMTGYVATRWYRAPE 25hP38b

DFGLARQADEEMTGYVATRWYRAPE 25hp38g

DFGLARQADSEMTGYVVTRWYRAPE 25hp38d

DFGLARHADAEMTGYVVTRWYRAPE 25hJNK1

DFGLARTAGTSFMMTPYVVTRYYRAPE 27hJNK2

DFGLARTACTNFMMTPYVVTRYYRAPE 27hJNK3

DFGLARTAGTSFMMTPYVVTRYYRAPE 27hERK1

DFGLARIADPEHDHTGFLTEYVATRWYRAPE 31hERK2

DFGLARVADPDHDHTGFLTEYVATRWYRAPE 31hBMK1DFGMARGLCTSPAEHQYFMTEYVATRWYRAPE 32YHOG1

DFGLARIQDPQMTGYVSTRWYRAPE 25YSMK1

DFGLARGIHAGFFKCHS--TVQPHITNYVATRWYRAPE 36YMPK1

DFGLARGYSENPVENSQFLTEYVATRWYRAPE 32YKSS1

DFGLARCLASSSDSRETLVGFMTEYVATRWYRAPE 35YFUS3

DFGLARIIDESAADNSEPTGQQSGMTEYVATRWYRAPE 38domainVIIVIIILoop-12(T-Loop)sequenceofMAPkinasesConstructionofp38loop-12toERKlikestructure

p38...DFGLARHTDDEMTGYVATRWYRAPE...p38(E)...DFGLARHTDDEMTEYVATRWYRAPE...p38(6+)...DFGLARHTDDEHDHTGFMTGYVATRWYRAPE...p38(6+E)...DFGLARHTDDEHDHTGFMTEYVATRWYRAPE...p38(VAP)...DFGLARVADPEMTGYVATRWYRAPE...p38(DL)...DFGLARHTDDDLTGYVATRWYRAPE...p38(VAPD6+LE)...DFGLARVADPDHDHTGFLTEYVATRWYRAPE...ERK2...DFGLARVADPDHDHTGFLTEYVATRWYRAPE...MAPKMKKSubstratesLoop-12isakeystructuretodeterminetheselectionofsubstrate(...TXY...)病毒技术对于揭示细胞信号通路的作用腺病毒逆转录病毒细胞信号分子特异性抑制剂的应用几种MAPK通路抑制剂的化学结构示意图:ONH2OCH3OPD98059HNNSOFCH3NOHNFNHNSB202190SB203580CH=CHCH=CHOHOCH3OHOCH3OOCurcuminEffectsofSB203580andPD98059onendogenousPRAKactivityHSP27TNF+++---------Arsenite+++---------PMA+++---------SB203580_---+--+---+PD98059----+--+--+-细胞信号分子的荧光标记LPSEGFControlp38p38p38p38p38p38p38p38LPS、UV刺激心肌细胞共聚焦显微镜大体扫描LPSControlUV细胞信号分子的三维结构分析蛋白质三维结构分析对于揭示信号分子的功能的作用蛋白质与蛋白质相互作用的研究PHTHSH3SH2KinaseBtkSH3SH2KinaseSrc蛋白激酶结构域1、蛋白质结合实验2、免疫共沉淀实验2、FRET和BRET与信号分子相互作用蛋白质分子的相互作用酵母双杂合系统IdentificationofMEF2Casasubstrateforp38第一步附着第二步激活第三步紧密粘附第四步渗出ECEC：趋化因子受体：PECAM：白细胞整合素：选择素：选择素配体：白细胞激活物：Ig家族成员图12.1白细胞的渗出过程

噬菌体展示技术报告基因技术研究细胞信号转导通路通过转录因子对基因启动子转录活性的影响。051015RelativeluciferaseactivityControlLPSEGFLPS+FHPIp38isinvolvedintheenhancementofTNF-promotertransactivityInductionofc-JunthroughMEF2Cphosphorylationbyp38蛋白质与核酸相互作用技术研究细胞内蛋白质尤其是转录因子与特定核酸序列的相互作用。EMSA对细胞内信号分子的干预技术研究细胞内蛋白质尤其是转录因子与特定核酸序列的相互作用。反义核酸技术的应用RNAiRNAiFigure1.Effectsof

mex-3RNAinterferenceonlevelsoftheendogenousmRNA.NomarskiDICmicrographsshowinsituhybridizationof4-cellstageembryos.(A)Negativecontrolshowinglackofstainingintheabsenceofthehybridizationprobe.(B)Embryofromuninjectedparentshowingnormalpatternofendogenous

mex-3RNA(purplestaining).(C)Embryofromparentinjectedwithpurified

mex-3antisenseRNA.Theseembryos(andtheparentanimals)retain

mex-3mRNA,althoughlevelsmaybesomewhatlessthanwildtype.(D)Late4-cellstageembryofromaparentinjectedwithdsRNAcorrespondingto

mex-3;no

mex-3RNAisdetected.(TemplatesusedforinterferingRNAandinsituprobeswerelargelynon-overlapping.)

信号分子基因表达检测技术研究细胞内mRNA表达水平。HeartBrainPlacentaLungLiverSkeletalMuscleKidneyPancreasp