>
产品中心 >
Other_kits >
AAT Bioquest/Amplite™ Colorimetric Beta-Lactamase Activity Assay Kit/12551/200 Tests
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 490/None |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | CellBIOLOGy ReporterGeneEnzymes |
| Related | HydrolyticEnzymes |
1.Prepareβ-lactamasestandardstocksolution:
Add100µLofddH2Ointothevialofβ-lactamasestandard(ComponentC)tomake50mU/mLβ-lactamasestandardstocksolution.
Note:Theunusedβ-lactamasestandardstocksolutionshouldbedividedintosingleusealiquotsandstoredat-20ºC.
2.Prepareserialdilutionsofβ-lactamasestandard:
2.1 Add10μLofβ-lactamasestandardstocksolution(50mU/mL,fromStep1)into990µL1×PBSbuffertogenerate500µU/mLβ-lactamasestandardsolution.
Note:Dilutedβ-lactamasestandardsolutionisunstable,andshouldbeusedpromptly.
2.2 Take200μLof500µU/mLβ-lactamasestandardsolutiontoperform1:2serialdilutionsinPBStogetapproximately250,125,62.5,31.3,15.6,7.8and0μU/mLserialdilutionsofβ-lactamasestandard.
2.3 Addserialdilutionsofβ-lactamasestandardandβ-lactamasecontainingtestsamplesintoa96-wellclearbottommicroplateasdescribedinTables1and2.
Table1Layoutofβ-lactamasestandardsandtestsamplesina96-wellclearbottommicroplate
BL | BL | TS | TS | …. | …. |
|
|
|
|
|
|
Lac1 | Lac1 | …. | …. | …. | …. |
|
|
|
|
|
|
Lac2 | Lac2 |
|
|
|
|
|
|
|
|
|
|
Lac3 | Lac3 |
|
|
|
|
|
|
|
|
|
|
Lac4 | Lac4 |
|
|
|
|
|
|
|
|
|
|
Lac5 | Lac5 |
|
|
|
|
|
|
|
|
|
|
Lac6 | Lac6 |
|
|
|
|
|
|
|
|
|
|
Lac7 | Lac7 |
|
|
|
|
|
|
|
|
|
|
Note:Lac=β-LactamaseStandards,BL=BlankControl,TS=TestSamples.
Table2Reagentcompositionforeachwell
β-LactamaseStandard | BlankControl | TestSample |
SerialDilutions*:50μL | 1×PBSBuffer:50μL | 50μL |
*Note:Addtheseriallydilutedβ-lactamasestandardsfromapproximately500to7.8μU/mLintowellsfromLac1to Lac7induplicate.
3.Prepareβ-lactamaseassaymixture:
Add50µLnitrocefinstocksolution(ComponentA)into5mLofComponentB,andmixwelltomakeβ-lactamaseassaymixture(ComponentA+B).
Note:Thisβ-lactamaseassaymixtureisenoughforone96-wellplate.Theβ-lactamaseassaymixtureisnotstable,preparefreshforeachuse.
4.Runβ-lactamaseassay:
4.1 Add50μLofβ-lactamaseassaymixture(fromStep3)toeachwellofβ-lactamasestandard,blankcontrol,andtestsamples(seeStep2.3)tomakethetotalvolumeof100µL/well.
Note:Fora384-wellplate,add25μLofsampleand25μLofβ-lactamaseassaymixtureintoeachwell.
4.2 Incubatethereactionatroomtemperaturefor30-60minutes,protectedfromlight.
4.3 MonitortheabsorbanceincreasewithanabsorbanceplatereaderatODratioof490/380nm.
| References&Citations |  PrinterFriendlyVersion |
- ChantemesseB,BetelliL,SolanasS,VienneyF,BollacheL,HartmannA,RocheletM.(2017)Anitrocefin-basedamperometricassayfortherapidquantificationofextended-spectrumbeta-lactamase-producingEscherichiacoliinwastewaters.WaterRes,109,375.
- RocheletM,SolanasS,BetelliL,NeuwirthC,VienneyF,HartmannA.(2015)Amperometricdetectionofextended-spectrumbeta-lactamaseactivity:applicationtothecharacterizationofresistantE.colistrains.Analyst,140,3551.
- HandaD,PandeyA,AsthanaAK,RawatA,HandaS,ThakuriaB.(2013)EvaluationofphenotypictestsforthedetectionofAmpCbeta-lactamaseinclinicalisolatesofEscherichiacoli.IndianJPatholMicrobiol,56,135.
- KarsisiotisAI,DamblonC,RobertsGC.(2014)Complete(1)H,(1)(5)N,and(1)(3)CresonanceassignmentsofBacilluscereusmetallo-beta-lactamaseanditscomplexwiththeinhibitorR-thiomandelicacid.BiomolNMRAssign,8,313.
- ThomasPW,SpicerT,CammarataM,BrodbeltJS,HodderP,FastW.(2013)Analteredzinc-bindingsiteconfersresistancetoacovalentinactivatorofNewDelhimetallo-beta-lactamase-1(NDM-1)discoveredbyhigh-throughputscreening.BioorgMedChem,21,3138.
- PriyadharsiniRI,KavithaA,RajanR,MathaviS,RajeshKR.(2011)Prevalenceofbla(CTXM)extendedspectrumbetalactamasegeneinenterobacteriaceaefromcriticalcarepatients.JLabPhysicians,3,80.
- VaidyaVK.(2011)HorizontalTransferofAntimicrobialResistancebyExtended-SpectrumbetaLactamase-ProducingEnterobacteriaceae.JLabPhysicians,3,37.
- FarmerR,GautamB,SinghS,YadavPK,JainPA.(2010)VirtualscreeningofAmpC/beta-lactamaseastargetforantimicrobialresistanceinPseudomonasaeruginosa.Bioinformation,4,290.
- SanchezPA,ToneyJH,ThomasJD,BergerJM.(2009)AsensitivecoupledHPLC/electrospraymassspectrometryassayforSPM-1metallo-beta-lactamaseinhibitors.AssayDrugDevTechnol,7,170.
- YuanJ,HuangW,ChowDC,PalzkillT.(2009)Finemappingofthesequencerequirementsforbindingofbeta-lactamaseinhibitoryprotein(BLIP)toTEM-1beta-lactamaseusingageneticscreenforBLIPfunction.JMolBiol,389,401.
