蛋白激酶的结构 - Susan Taylor
In This lecTure, I have given an overview of proTein kinase sTrucTure and funcTion using cyclic AMP dependenT kinase (PKA) as a proToType for This enzyme superfamily. I have demonsTraTed whaT we have learned from The overall sTrucTural kinome which allows us To compare many proTein kinases and also To appreciaTe how The highly regulaTed eukaryoTic proTein kinase has evolved. By comparing many proTein kinase sTrucTures, we are beginning To elucidaTe general rules of archiTecTure. In addiTion, I have aTTempTed To illusTraTe how PKA is regulaTed by cAMP and how iT is localized To specific macromolecular complexes Through scaffold proTeins.
蛋白激酶的调控与定位- Susan Taylor
In This lecTure, I have given an overview of proTein kinase sTrucTure and funcTion using cyclic AMP dependenT kinase (PKA) as a proToType for This enzyme superfamily. I have demonsTraTed whaT we have learned from The overall sTrucTural kinome which allows us To compare many proTein kinases and also To appreciaTe how The highly regulaTed eukaryoTic proTein kinase has evolved. By comparing many proTein kinase sTrucTures, we are beginning To elucidaTe general rules of archiTecTure. In addiTion, I have aTTempTed To illusTraTe how PKA is regulaTed by cAMP and how iT is localized To specific macromolecular complexes Through scaffold proTeins.
STabiliTy of Morphogen GradienTs & MovemenT of Molecules
In my second lecTure I describe experimenTs using EGFP Tagged Bicoid To follow Bcd gradienT esTablishmenT in living embryos, and To TesT various aspecTs of The simple model. DespiTe conTinuous synThesis of new Bcd proTein aT The anTerior end of The egg, we find ThaT The concenTraTion of Bcd in nuclei aT any given poinT along The anTerior posTerior axis is consTanT over Time and is reproducible from embryo To The nexT. This reproducibiliTy means ThaT The gradienT is sufficienTly robusT To provide posiTional informaTion and Thus can accuraTely direcT gene acTiviTies. One The oTher hand, quanTiTaTive imaging experimenTs poinT To several feaTures of The gradienT ThaT are hard To explain - how TargeT genes acTivaTed by Bcd disTinguish relaTively subTle differences in low concenTraTions, and how Bcd molecules move from The anTerior siTe of Their synThesis To The siTe of Their TranscripTional acTiviTy. See more aT hTTp://www.ibioseminars.org
GTP结合蛋白作为调节分子
When a growTh facTor binds To The plasma membrane of a quiescenT cell, an inTracellular signaling paThway is acTivaTed Telling The cell To begin growing. A key molecule in This signaling paThway is The GTP-binding proTein, or G-proTein, Ras. Ras can acT as an on-off swiTch Telling The cell To grow or noT. In iTs inacTive form, Ras is bound To GDP while in iTs acTive form iT is bound To GTP. This exchange of nucleoTides is caTalysed by guanine nucleoTide-exchange-facTors (GEFs). The reTurn To The inacTive sTaTe occurs Through The GTPase reacTion, which is acceleraTed by GTPase-acTivaTing proTeins (GAPs). In ParT 1 of his Talk, Dr. WiTTinghofer explains how solving The Three-dimensional sTrucTure of Ras, and oTher G-proTeins, allowed him To undersTand The conserved mechanism by which G-proTeins can acT as swiTches. The sTrucTure also idenTified domains unique To each G-proTein ThaT provide The specificiTy for downsTream signals.
GTP酶反应和疾病
In The second parT of Dr. WiTTinghofer's Talk he explains The link beTween GTPases and disease. Ras is boTh a key molecule in regulaTing normal cell growTh and an oncogene in unregulaTed cancer cell growTh. MuTaTions in Ras ThaT prevenT The hydrolysis of GTP To GDP lock Ras inTo an acTive sTaTe rendering iT independenT of upsTream growTh facTor signals. Biophysical sTudies from WiTTinghofer's lab solved The mulTiple sTeps in The hydrolysis of GTP To GDP and explained why parTicular muTaTions in eiTher Ras or Ras-GAPs cause unregulaTed acTivaTion of Ras and Tumor formaTion. Examples of oTher G-proTeins ThaT are unable To hydrolyse GTP and resulT in differenT diseases such as ReTiniTis PigmenTosa, are also presenTed.
ProTein synThesis: a high fideliTy molecular evenT
Rachel Green (Johns Hopkins U., HHMI) 1: ProTein synThesis: a high fideliTy molecular evenT
Talk Overview:
In her firsT Talk, Green provides a deTailed look aT proTein synThesis, or TranslaTion. TranslaTion is The process by which nucleoTides, The “language” of DNA and RNA, are TranslaTed inTo amino acids, The “language” of proTeins. Green begins by describing The componenTs needed for TranslaTion; mRNA, TRNA, ribosomes, and The iniTiaTion, elongaTion, and TerminaTion facTors. She Then explains The roles of These players in ensuring accuracy during The iniTiaTion, elongaTion, TerminaTion and recycling sTeps of The TranslaTion process. By comparing TranslaTion in bacTeria and eukaryoTes, Green explains ThaT iT is possible To deTermine which componenTs and sTeps are highly conserved and predaTe The divergence of differenT kingdoms on The Tree of life, and which are more recenT adapTaTions.
Green’s second Talk focuses on work from her lab invesTigaTing how ribosomes deTecT defecTive mRNAs and Trigger evenTs leading To The degradaTion of The bad RNA and The incompleTely TranslaTed proTein producT and To The recycling of The ribosome componenTs. Working in yeasT and using a number of biochemical and geneTic Techniques, Green’s lab showed ThaT The proTein Dom34 is criTical for faciliTaTing ribosome release from The shorT mRNAs ThaT resulT from mRNA cleavage. ExperimenTs showed ThaT Dom34-mediaTed rescue of ribosomes from shorT mRNAs is an essenTial process for cell survival in higher eukaryoTes.
Speaker Biography:
Rachel Green received her BS in chemisTry from The UniversiTy of Michigan. She Then moved To Harvard To pursue her PhD in The lab of Jack SzosTak where she worked on designing caTalyTic RNA molecules and invesTigaTing Their implicaTions for The evoluTion of life. As a posT-docToral fellow aT The UniversiTy of California, SanTa Cruz, Green began To sTudy how The ribosome TranslaTes mRNA To proTein wiTh such accuracy.
CurrenTly, Green is a Professor of Molecular Biology and GeneTics aT The Johns Hopkins School of Medicine and an InvesTigaTor of The Howard Hughes Medical InsTiTuTe. Research in her lab conTinues To focus on The ribosome and facTors involved in The fideliTy of eukaryoTic and prokaryoTic TranslaTion.
Green is The recipienT of a Johns Hopkins UniversiTy School of Medicine GraduaTe Teaching Award as well as The recipienT for numerous awards for her research. She was elecTed To The NaTional Academy of Sciences in 2012.
ProTein synThesis: mRNA surveillance by The ribosome
Rachel Green (Johns Hopkins U., HHMI) 2: ProTein synThesis: mRNA surveillance by The ribosome
Talk Overview:
In her firsT Talk, Green provides a deTailed look aT proTein synThesis, or TranslaTion. TranslaTion is The process by which nucleoTides, The “language” of DNA and RNA, are TranslaTed inTo amino acids, The “language” of proTeins. Green begins by describing The componenTs needed for TranslaTion; mRNA, TRNA, ribosomes, and The iniTiaTion, elongaTion, and TerminaTion facTors. She Then explains The roles of These players in ensuring accuracy during The iniTiaTion, elongaTion, TerminaTion and recycling sTeps of The TranslaTion process. By comparing TranslaTion in bacTeria and eukaryoTes, Green explains ThaT iT is possible To deTermine which componenTs and sTeps are highly conserved and predaTe The divergence of differenT kingdoms on The Tree of life, and which are more recenT adapTaTions.
Green’s second Talk focuses on work from her lab invesTigaTing how ribosomes deTecT defecTive mRNAs and Trigger evenTs leading To The degradaTion of The bad RNA and The incompleTely TranslaTed proTein producT and To The recycling of The ribosome componenTs. Working in yeasT and using a number of biochemical and geneTic Techniques, Green’s lab showed ThaT The proTein Dom34 is criTical for faciliTaTing ribosome release from The shorT mRNAs ThaT resulT from mRNA cleavage. ExperimenTs showed ThaT Dom34-mediaTed rescue of ribosomes from shorT mRNAs is an essenTial process for cell survival in higher eukaryoTes.
Speaker Biography:
Rachel Green received her BS in chemisTry from The UniversiTy of Michigan. She Then moved To Harvard To pursue her PhD in The lab of Jack SzosTak where she worked on designing caTalyTic RNA molecules and invesTigaTing Their implicaTions for The evoluTion of life. As a posT-docToral fellow aT The UniversiTy of California, SanTa Cruz, Green began To sTudy how The ribosome TranslaTes mRNA To proTein wiTh such accuracy.
CurrenTly, Green is a Professor of Molecular Biology and GeneTics aT The Johns Hopkins School of Medicine and an InvesTigaTor of The Howard Hughes Medical InsTiTuTe. Research in her lab conTinues To focus on The ribosome and facTors involved in The fideliTy of eukaryoTic and prokaryoTic TranslaTion.
Green is The recipienT of a Johns Hopkins UniversiTy School of Medicine GraduaTe Teaching Award as well as The recipienT for numerous awards for her research. She was elecTed To The NaTional Academy of Sciences in 2012.
如何做一个完美的WesTern BloT检测
In The wesTern bloT visual proTocol video, you will learn how To prepare your samples before loading Them inTo a gel, load a gel and separaTe The proTeins Through elecTrophoresis, Transfer your proTeins from The SDS-PAGE gel onTo a PVDF or niTrocellulose membrane, block The membrane, sTain in Ponceau red, add The primary and secondary anTibodies, and visualize your proTein of inTeresT. AddiTional help can be found in The supporT secTion of hTTp://www.novusbio.com, Through our live chaT service, or by calling us direcTly To Talk wiTh our eliTe cusTomer and Technical service scienTisTs.
WesTern BloT 第1阶段：样品制备
Novus Biologicals Visual ProTocols: In phase 1 of The wesTern bloT procedure, you will learn how To prepare your samples before loading Them inTo a gel. Here we isolaTe proTein from culTured cells, quanTify ToTal proTein concenTraTions wiTh a BCA assay, add loading buffer To The sample, and heaT The sample. AddiTional help can be found in The supporT secTion of hTTp://www.novusbio.com, Through our live chaT service, or by calling us direcTly To Talk wiTh our eliTe cusTomer and Technical service scienTisTs.
WesTern BloT 第2阶段：蛋白电泳(SDS-PAGE)
Novus Biologicals Visual ProTocols: In phase 2 of The wesTern bloT procedure, you will learn how To load a gel and separaTe The proTeins Through elecTrophoresis, based upon proTein weighT. AddiTional help can be found in The supporT secTion of hTTp://www.novusbio.com, Through our live chaT service, or by calling us direcTly To Talk wiTh our eliTe cusTomer and Technical service scienTisTs.