| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 650/None |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | SmallMoleculeDetection DiagnosticMolecules |
| Related | CellMetabolism BiochemicalAssays |
1. PrepareserialdilutionsofAmmoniumChloride(0to1mM)solutions:
1.1 Add1μLof1.0MAmmoniumChlorideStandard(ComponentC)to999µLDPBStogenerate1.0mMstandard
ammoniumchloridesolution.
1.2 Take300μLof1.0mMstandardtoperform1:3serialdilutionstoget300,100,30,10,3,1,and0µMstandard
ammoniumchloridesolutions.
1.3 AddAmmoniumChlorideStandardsandammoniacontainingtestsamplesintoa96-wellclearbottommicroplateasdescribedinTables1and2.
Table1.Layoutofstandardsandtestsamplesinaclearbottom96-wellmicroplate:
BL | BL | TS | TS | …. | …. |
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AS1 | AS1 | …. | …. | …. | …. |
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AS2 | AS2 |
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AS3 | AS3 |
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AS4 | AS4 |
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AS5 | AS5 |
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AS6 | AS6 |
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AS7 | AS7 |
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Note:AS=AmmoniumChlorideStandards,BL=BlankControl,TS=TestSamples.
Table2.Reagentcompositionforeachwell:
AmmoniumChlorideStandard | BlankControl | TestSample |
Serialdilutions*:50μL | DPBS:50μL | 50μL |
*Note:Addtheseriallydilutedammoniumchloridestandardsfrom1to1000µMintowellsfromAS1toAS7induplicate.
2. RunAmmoniumChlorideAssay:
2.1 Add50μLofAssayBufferI(ComponentA)toeachwelloftheAmmoniumChlorideStandard,blankcontrol,andtestsamples(seeStep1.3)sothatthetotalassayvolumeis100µL/well.
Note:Fora384-wellplate,add25μLsample,25μLofAssayBufferIperwell.
2.2 Incubatethereactionfor5minutesatroomtemperature.
2.3 Add50μLofAssayBufferII(ComponentB)toeachwellsothatthetotalassayvolumeis150µL/well.
Note:Fora384-wellplate,add25μLAssayBufferII(ComponentB)toeachwell.
2.4 Incubateatroomtemperaturefor30-60minutes,andmonitortheabsorbanceincreaseat660-670nmusinganabsorbancemicroplatereader.
Note:ThecolorturnstoyellowafterAssayBufferII(ComponentB)isadded,andthewellswithAmmoniumChlorideStandardorsampleswillshowbluishgreencolorafterincubation.Theintensityofthecolorwillreachthemaximumin30-60minutes.
| References&Citations |  PrinterFriendlyVersion |
1. LohTP,TanKM,ChewS,ChanDS.(2013)Highpleuralammonianegativelyinterfereswiththemeasurementofadenosinedeaminaseactivity.BMJCaseRep,2013.
2. BakLK,WaagepetersenHS,SorensenM,OttP,VilstrupH,KeidingS,SchousboeA.(2013)Roleofbranchedchainaminoacidsincerebralammoniahomeostasisrelatedtohepaticencephalopathy.MetabBrainDis.
3. TranahTH,VijayGK,RyanJM,ShawcrossDL.(2013)Systemicinflammationandammoniainhepaticencephalopathy.MetabBrainDis,28,1.
4. HenryRP,LucuC,OnkenH,WeihrauchD.(2012)Multiplefunctionsofthecrustaceangill:osmotic/ionicregulation,acid-basebalance,ammoniaexcretion,andbioaccumulationoftoxicmetals.FrontPhysiol,3,431.
5. RothmanDL,DeFeyterHM,MaciejewskiPK,BeharKL.(2012)Isthereinvivoevidenceforaminoacidshuttlescarryingammoniafromneuronstoastrocytes?NeuRochemRes,37,2597.
6. StahlDA,delaTorreJR.(2012)Physiologyanddiversityofammonia-oxidizingarchaea.AnnuRevMicrobiol,66,83.
7. ShenJP,ZhangLM,DiHJ,HeJZ.(2012)Areviewofammonia-oxidizingbacteriaandarchaeainChinesesoils.FrontMicrobiol,3,296.
8. WrightPA,WoodCM.(2012)Seventhingsfishknowaboutammoniaandwedon"t.RespirPhysiolNeurobiol,184,231.
9. RoseCF.(2012)Ammonia-loweringstrategiesforthetreatmentofhepaticencephalopathy.ClinPharmacolTher,92,321.
10. deVilliersM,PuthanVeetilV,RajH,deVilliersJ,PoelarendsGJ.(2012)Catalyticmechanismsandbiocatalyticapplicationsofaspartateandmethylaspartateammonialyases.ACSChemBiol,7,1618.
