method detects only free fluoride ions in solution Because of the inherent restriction of this technique, several approaches have been recommended to prepare the sample for analysis Lopez and Navia (1988) assayed total fluoride (bound and free) in food and beverages by initially acid hydrolyzing samples at 100 °C in borosilicate vials
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G-Biosciences 1-800-628-7730 1-314-991-6034 technical@GBiosciences.com
AGenoTechnology,Inc.(USA)brandname
think proteins! think G-Biosciences www.GBiosciences.com PR110
DotBlotAnalysis
Teacher'sGuidebook
(Cat. #BEǦ502) ........3 ...........3 ........3 TIME ........6 ..................7 ..............7 .............................8 ..........9
MATERIALSINCLUDEDWITHTHEKIT
each).
1vialSimulatedSample1
1vialSimulatedSample2
1vialIMUPositiveControl
1vialIMUNegativeControl
1vialAntigenBindingBuffer
1packProteinBindingMembraneStrips
1bottleBlockingBuffer(2XNAPͲBlocker)
1bottleMEMWashingBuffer(10X)
1vialAntibody:BEAntibody1(Ab:BEͲ1)
1bottleHRPSubstrate
30
CentrifugeTubes(1.5ml)
1tubeSterileWater
SPECIALHANDLINGINSTRUCTIONS
ADDITIONALEQUIPMENTREQUIRED
ShakingIncubator
PlasticWashingTrays12cmx12cm
TIMEREQUIRED
4Ͳ6hours
Page3of12
OBJECTIVES
TounderstandtheprincipleofDotBlotting.
UseofDotblottingfordiagnostictests.
INTRODUCTION
Dot diagnostictool. phosphatase(AP). An placed
Page4of12
HowAreAntibodiesMade(PrimaryAntibody)?
Enzyme
LabeledAntibodies(SecondaryAntibodies)
Dot detected. tetramethylbenzidine(TMB). allowingthemtoidentifyinfectedpatients.
Page5of12
TEACHER'SPREǦEXPERIMENTSETUP
1. Allowallreagentsstoredinthecoldtowarmtoroomtemperature.
2. Add200µlofAntigenBindingBuffertoeachvialoflyophilizedantigen(Simulated
Mixwellbyvortexing.
3. Transfer20lSimulatedSample2toacleantubeandadd180lAntigenBinding
experiments.
4. Labelsixsetsof4tubeseitherP1,P2,positive,ornegativeforpatient1,patient2,
NegativeControl.
5. Add250µlofsterilewatertothelyophilizedprimaryandsecondaryantibody
groupwith1vial.
6. Makea1XMEMWashingBuffersolutionbyadding200mlMEMWashingBuffer
detergenttoaidinmembranewashing.
7. Themorningoftheexperiment,gentlyshakethesuppliedBlockingBuffer(2XNAPͲ
WashingBuffer.
Page6of12
MATERIALSFOREACHGROUP
IMUNegativeControl
1XMEMWashingBuffer(sharedwithclass)
1vialAntibody:BEAntibody1
1vialAntibody:BEAntibody4(HRPSecondary)
1bottleofHRPSubstrate(sharedwithclass)
4stripsofProteinBindingMembrane
250mLtubes
112cmx12cmwashingtray
PROCEDURE
SpottingSampleProtocol
1. Usingforcepsremovethenitrocellulosemembranestripfromtheprotectivecover.
2. Usinga5Ͳ10lpipette,remove5lofthesampleandspotontothemembrane.
samplesonthestripasinthefigurebelow.
3. Onceallstudentsinyourgroupfinishedspottingandallliquidhasabsorbedonto
Page7of12
ProteinDetection
1. Add20ml1XBlockingBuffer(NAPͲBlocker)toblockthenonͲspecificsites.Incubate
2. Preparetheprimaryantibodybyadding40lBEAntibody1to20ml1XBlocking
Buffer(NAPͲBlocker).
3. DiscardtheBlockingBuffer.Addtheprimaryantibodysolutiontothemembrane
4. Discardtheantibodysolutionandwash3timeswith20mlMEMWashingBufferfor
10 minuteseach.
5. Makesecondaryantibodybymixing40µlBEAntibody4(HRPSecondary)with20ml
horseradishperoxidasetag.
6. DiscardtheMEMWashingBufferandaddthesecondaryantibodysolutiontothe
membrane shaking.
7. Discardtheantibodysolutionandwash3timeswith20ml1XMEMWashingBuffer
for10minuteseach.
8. DiscardtheMEMWashingBufferandadd5mlofHRPSubstratetothemembrane.
9. PouroffthesubstrateandaddDIwatertostopthecolorreaction.Recordyour
results.
Page8of12
Page9of12RESULTS,ANALYSIS&ASSESSMENT
1. Whichofthepatientsiscarryingthehighestlevelofinfection?
Patient
2. DescribethecircumstancesinwhichonlyoneantibodyisrequiredforDotblotting.
secondaryantibody
3. Whatisthefunctionoftheblockingstep?
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Page10of12
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Page11of12
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Page12of12
G-Biosciences 1-800-628-7730 1-314-991-6034 technical@GBiosciences.com
AGenoTechnology,Inc.(USA)brandname
think proteins! think G-Biosciences www.GBiosciences.com PR111
DotBlotAnalysis
Student'sHandbook
(Cat. #BEǦ502) ..................5 ..............5 .............................6 ..........7
OBJECTIVES
TounderstandtheprincipleofDotBlotting.
UseofDotblottingfordiagnostictests.
INTRODUCTION
Dot diagnostictool. phosphatase(AP). An placed
Page3of8
serum
HowAreAntibodiesMade(PrimaryAntibody)?
Enzyme
LabeledAntibodies(SecondaryAntibodies)
Dot detected. tetramethylbenzidine(TMB). allowingthemtoidentifyinfectedpatients.
Page4of8
MATERIALSFOREACHGROUP
IMUNegativeControl
1XMEMWashingBuffer(sharedwithclass)
1vialAntibody:BEAntibody1
1vialAntibody:BEAntibody4(HRPSecondary)
1bottleofHRPSubstrate(sharedwithclass)
4stripsofProteinBindingMembrane
250mLtubes
112cmx12cmwashingtray
PROCEDURE
SpottingSampleProtocol
1. Usingforcepsremovethenitrocellulosemembranestripfromtheprotectivecover.
2. Usinga5Ͳ10lpipette,remove5lofthesampleandspotontothemembrane.
samplesonthestripasinthefigurebelow.
3. Onceallstudentsinyourgroupfinishedspottingandallliquidhasabsorbedonto
Page5of8
ProteinDetection
1. Add20ml1XBlockingBuffer(NAPͲBlocker)toblockthenonͲspecificsites.Incubate
2. Preparetheprimaryantibodybyadding40lBEAntibody1to20ml1XBlocking
Buffer(NAPͲBlocker).
3. DiscardtheBlockingBuffer.Addtheprimaryantibodysolutiontothemembrane
4. Discardtheantibodysolutionandwash3timeswith20mlMEMWashingBufferfor
10 minuteseach.
5. Makesecondaryantibodybymixing40µlBEAntibody4(HRPSecondary)with20ml
horseradishperoxidasetag.
6. DiscardtheMEMWashingBufferandaddthesecondaryantibodysolutiontothe
membrane shaking.
7. Discardtheantibodysolutionandwash3timeswith20ml1XMEMWashingBuffer
for10minuteseach.
8. DiscardtheMEMWashingBufferandadd5mlofHRPSubstratetothemembrane.
9. PouroffthesubstrateandaddDIwatertostopthecolorreaction.Recordyour
results.
Page6of8
Page7of8RESULTS,ANALYSIS&ASSESSMENT
1. Whichofthepatientsiscarryingthehighestlevelofinfection?
2. DescribethecircumstancesinwhichonlyoneantibodyisrequiredforDotblotting.
3. Whatisthefunctionoftheblockingstep?
Lastsaved:8/30/2012CMH
www.GBiosciences.com
Page8of8
quotesdbs_dbs18.pdfusesText_24