胞外基质
来自 : 蚂蚁淘
- ECMCellAttachmentAssay(LTI)
- CellAdherenceInhibitionAssay(LTI)Generalprotocol--EithermonoclonalantibodyorRGDpeptideisaddedalongwiththecellsduringthestandardadhesionassay,andtheinhibitoryeffectcanthusbesemi-quantitated.
- ECMCellELISA(LTI)Thisisgeneralprotocol.Specificantibodiestointegrinsareaddedtopelletedcellsandallowedtobindtothecellsurface.Thenonboundantibodyiswashedawayandanenzymeconjugatedsecondantibodyisaddedtothere-pelletedcells.Theboundsecondantibodyisthendetectedbytheadditionofanenzyme-specificsubstrateandtheplateortubesarereadspectrophotometrically.Thisassaycanbeusedasascreeningassayforallintegrinsforwhichantibodiesareavailable.
- ECMDirectELISA(LTI)Thisprocedurecanbeusedfordetectionofspecificproteinsorforthetitrationofanantibody.AproteinormixtureofproteinsiscoatedonapolystyreneELISAplate,andantibodyisallowedtobindtotheprotein.Theboundantibodyisdetectedbytheadditionofasecondantibodyconjugatedtoanenzymeandanenzymesubstratewithcolordetectorisadded.Therelativeintensityofthecolordevelopedisproportionaltotheoriginalconcentrationofproteincoatedontheplateandtotheconcentrationofantibodyboundtocoatedantigen.
- ECMImmunoprecipitation(LTI)Integrinreceptorsoncellsurfacesarelabeledwithbiotin;thereceptorsarethenextractedfromthemembranesbydetergenttreatmentandsavedforimmunoprecipitation.Integrin-specificantibodiesareboundtoSepharosebeadscoatedwithanti-IgGandaddedtothebiotinylatedcellextract.BoundreceptorsarerunonanSDS-PAGE,transferredtonitrocellulose,anddetectedbytheadditionofstreptavidin-enzymeconjugatefollowedbysubstrate.
- ECMProtocolsWesternBlot(LTI)AmixtureofproteinisseparatedelectrophoreticallybySDS-PAGE,andtheindividualproteinbandsaretransferredtonitrocellulosepaper.Specificantibodyisthenusedtoprobeforanyofthebandsitmightbindto.Theboundantibodyisthendetectedbytheadditionofasecondantibodyagainsttheimmunoglobulinspeciescontainedinthefirstantibody.Thissecondantibodyislabeledwithanenzyme,andthespecificproteinbandcanthenbevisualizedbytheadditionofanenzymesubstratecontainingacolordeveloper.
- Fibrinogen-Purification(TheCellBIOLOGyandCytoskeletonGroup,HMS)
- IsolationofFishFibronectinfromFishFibrinogen(TheCellBiologyandCytoskeletonGroup,HMS)
- PlasminogenIsolation(TheCellBiologyandCytoskeletonGroup,HMS)
- Prothrombin-Activation(TheCellBiologyandCytoskeletonGroup,HMS)
- Prothrombin-Purification(TheCellBiologyandCytoskeletonGroup,HMS)
- Thrombin-PreparationfromFish(TheCellBiologyandCytoskeletonGroup,HMS)
- "CellsonGels"(TheCellBiologyandCytoskeletonGroup,HMS)Thisprotocoldescribesmethodforculturecellonathinpolyacrylamide-based,collagen-coatedflexIBLesubstrate.Bymaintainingaconstanttotalconcentrationofacrylamidewhileusr/localyingtheconcentrationofbis-acrylamide,it"sabletoobtainaseriesofchemicallyidenticalsubstrateswithawiderangeofflexibility.Byusingimagingtechniques,cells"responsetodifferencesinsubstrateflexibilitycanbedetected.
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