106

In vitro (Subject) / Zellbio (Lesson)

In vitro modelle

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  • PPI detection :protein-protein interaction Tango: transient interactions; ligand-receptor interactions; live cells; druggable human GPCRs B/FRET: live cells; photobleaching, background fluor (disadvantages) LUMIER: FLAG-tagged; cell lysis required (disadvantages) MYTH: halves of ubiquitin; live cells, bait can be overexpressed PCA: protein-fragment complementation assay; live, high throughput
  • kinase assay immunochemical kinase assay: determine activities of kinases; synthetic substrate peptide + mAb(recog phospho peptide), detection by enzymatic reaktion or light emission; performed on crude cell extracts.. enhanced by microfluidic PepArray TRF & FRET: DELFIA, Alpha screen In cell western: phosph percentage gegen inhibitor conc; cultured target cells > kinase inhibitor dilutions, stimulation > fix and permeablize cells, wash and block > rabbit anti-pan protein antibody > goat anti-rabbit                                                                                             or> mouse anti-phosphoantibody > goat anti-mouse
  • Ca regulation (RTK) indicator ligand regulated ca channel: in ER: IP3 receptor channel voltage regu Ca-ATPase Na/Ca-antiport indicators: Ca-binding fluor dyes (Fluo-4); Cameleon(tandem fusion of BFP or CFP, calmodulin, M13, GFP or YFP); Ionophores
  • regulatory functions of scaffold proteins in signaling cascades large multidomain proteins with a lot of structural disorder, able to regulate several members of a signaling pathway functions: assemble components of signaling pathway;                localize .. to s specific intracellular compartment or location;                regulate positive or nega feedback signals;                protect active signaling intermediate proteins from deactivation by phosphatases example: kinase suppressor of RAS (KSR), ERK pathway
  • MAPK feedback RAF-MEK-ERK cascade and feedback from ERK to RAF depending on feedback topologies and jinetic parameters> diff temporal responses: monotone, sustained response; transient, adaptive res; damped oscillations; sustained oci; switch-like, bistable response (on and off) negative feedback: increase resistance to disturbances inside the feedback loop; positive: increase sensitivity to the signal 
  • feedforward loop coherent feedforward loop: both branches are stimulatory or inhibitory incoherent: one stimu another inhibi
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