• 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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