[PDF] Dot BlotAnalysis - G-Biosciences



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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?

Lastsaved:8/30/2012CMH

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Page10of12

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Page11of12

www.GBiosciences.com

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

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