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Workflow Analysis for the Northeast Structural Genomics
Consortium at the CABM/Rutgers University/RWJMS
Protein Production Facility
October 22, 2002 Celia BiamonteSeptember 10,2005 Michael Baran
1Cloning &
Small Scale Expression
1Cloning &
Small Scale Expression
2Protein
Expression
2Protein
Expression
3Protein
Purification(Ni-NTAAffinity
Column)
3Protein
Purification(Ni-NTAAffinity
Column)
4HSQC
Screening
4HSQC
Screening
5Analytical
Gel Filtration/Dynamic
LightScattering
5Analytical
Gel Filtration/Dynamic
LightScattering
6Preparative GelFiltration underMonodisperse
Conditions
6Preparative GelFiltration underMonodisperse
Conditions
7Deliver Samples
For Crystallographic
Screening
7Deliver Samples
For Crystallographic
Screening
8Manufacture
EnrichedProtein forStructural
Studies
8Manufacture
EnrichedProtein forStructural
Studies
9Deliver
EnrichedSamples
For StructureDetermination
9Deliver
EnrichedSamples
For StructureDetermination
GoodResults
yes
no
Stop work
NESG Workflow AnalysisLevel 0 – Process Map
GoodResults
yesNoStop Work
1. Cloning & Small Scale Expression – Input/Output Model
1Cloning &
Small Scale Expression
1Cloning &
Small Scale Expression
Inputs•NESG Targets
Outputs•NESG Target List•Primers•Primer Design Spreadsheet•Cloned Targets•Cloning Chart•Gel Pictures of Solubility of each ORF•Laboratory Notebook
1. Cloning & Small Scale ExpressionLevel 1 – Process Map
1.1Procure NESG
Target List
1.1Procure NESG
Target List
1.2Identify Targets
1.2Identify Targets
1.3Perform
PCR
1.3Perform
PCR
1.4Obtain Clone
1.4Obtain Clone
1.5Conduct Small-
Scale Expression
1.5Conduct Small-
Scale Expression
Inputs
Outputs
•SPINE/Zebaview
•NESG Target List•Primer Design Spreadsheet
•Primers•Data Control Sheet
•Purified Gel Slice•Qiagen Kit•Primer Design Spreadsheet
•Colonies•Ethanol-Precipitated DNA
1.1 Procure NESG Target ListLevel 2 – Process Map
1.1.1View NESG
Targets
1.1.1View NESG
Targets
1.1.2Execute PrimerPrimer
Program
1.1.2Execute PrimerPrimer
Program
1.1.3Cut & Paste
ResultsInto Excel
1.1.3Cut & Paste
ResultsInto Excel
Inputs
Outputs
1.2 Identify TargetsLevel 2 – Process Map
1.2.1Review NESG
Target List
1.2.1Review NESG
Target List
1.2.2Select
Organism
1.2.2Select
Organism
1.2.3Assign WorkTo Analyst
1.2.3Assign WorkTo Analyst
Inputs
Outputs
1.2.4Order
Primers
1.2.4Order
Primers
1.3 Perform PCRLevel 2 – Process Map
1.3.1PCR
AmplificationOf
ORF
1.3.1PCR
AmplificationOf
ORF
1.3.2Agarose Gel
Analysis
1.3.2Agarose Gel
Analysis
1.3.3ReRun PCR
If Nescessary
1.3.3ReRun PCR
If Nescessary
Inputs
Outputs
1.3.4Manually Reconcile
Target Name & well location
with target sizeand restriction
sites
1.3.4Manually Reconcile
Target Name & well location
with target sizeand restriction
sites
1.4 Obtain CloneLevel 2 – Process Map
1.4.1Perform
Gel Extraction
1.4.1Perform
Gel Extraction
1.4.2Perform
RestrictionDigests
1.4.2Perform
RestrictionDigests
1.4.3Purify DNA
1.4.3Purify DNA
Inputs
Outputs
1.4.4Order
Primers
1.4.4Order
Primers1.4.5
Transform
1.4.5Transform
1.4.6Perform Colony
PCR
1.4.6Perform Colony
PCR
1.4.7Perfrom
Mini-Prep
1.4.7Perfrom
Mini-Prep
1.4.8Transform
intoExpressionHost Cells
1.4.8Transform
intoExpressionHost Cells
1.5 Conduct Small Scale ExpressionLevel 2 – Process Map
1.5.1SelectSingle
Colony &Grow
1.5.1SelectSingle
Colony &Grow
1.5.2ChangeMedia
1.5.2ChangeMedia
1.5.3Innoculate
&Grow
overnight
1.5.3Innoculate
&Grow
overnight
Inputs
Outputs
1.5.4Innoculate
IntoNew
culture &GrowUntil
OD ~0.6
1.5.4Innoculate
IntoNew
culture &GrowUntil
OD ~0.6
1.5.5Induce
1.5.5Induce
1.5.6Harvest
&FreezePellet
1.5.6Harvest
&FreezePellet
1.5.7Sonicate
1.5.7Sonicate
1.5.8Perform
SDS-Page
1.5.8Perform
SDS-Page
1.5.10Send
SolubleORFsFor
Sequen-cing
1.5.10Send
SolubleORFsFor
Sequen-cing
1.5.9Check
For Solubility
1.5.9Check
For Solubility
2. Protein Expression – Input/Output Model
2Protein
Expression
2Protein
Expression
Inputs•Plasmid•Cloning Chart•Work Order FormFrom Tom Acton
Outputs•Expressed Protein inCells•Gel Pictures of ProteinExpression Level, Solubility and Optical density data•Laboratory Norebook
2. Protein ExpressionLevel 1 – Process Map
2.1ReviewSolubleORFs
2.1ReviewSolubleORFs
2.2Select &Prepare
Media
2.2Select &Prepare
Media
2.3Transform
Cells
2.3Transform
Cells
Inputs
Outputs
2.4Grow Cells
& Measure
OD
2.4Grow Cells
& Measure
OD
2.5Induce
Cells
2.5Induce
Cells
2.6Prepare
&FreezePellets
2.6Prepare
&FreezePellets
2.7Conduct
FermentationAnalysis
2.7Conduct
FermentationAnalysis
2.2A Select and Prepare Media – Target Molecular Weight > 25KD Level 2 – Process Map
2.2.1ACheck
Molecular Weight
2.2.1ACheck
Molecular Weight
2.2.2AExpress
InTB
2.2.2AExpress
InTB
2.2.3APrepareMedia
2.2.3APrepareMedia
Inputs
Outputs
2.2B Select and Prepare Media – Target Molecular Weight 15-25KD Level 2 – Process Map
2.2.1BCheck
Molecular Weight
2.2.1BCheck
Molecular Weight
2.2.2BExpress
In TB (1L),And MJ9 with
100% 15N(0.5L)
2.2.2BExpress
In TB (1L),And MJ9 with
100% 15N(0.5L)
2.2.3BPrepareMedia
2.2.3BPrepareMedia
Inputs
Outputs
2.2C Select and Prepare Media – Target Molecular Weight < 15KD Level 2 – Process Map
2.2.1CCheck
Molecular Weight
2.2.1CCheck
Molecular Weight
2.2.2CExpress
In MJ9 with100% 15N &
5% 13C
2.2.2CExpress
In MJ9 with100% 15N &
5% 13C
2.2.4CPrepareMedia
2.2.4CPrepareMedia
Inputs
Outputs
2.2.3CExpress
In TB
2.2.3CExpress
In TB
2.4 Grow Cells and Read ODLevel 2 – Process Map
2.4.1Grow 3mL ofTransformed
Cells inSmall Tube
2.4.1Grow 3mL ofTransformed
Cells inSmall Tube
2.4.2Grow Cells to
25 mLOvernight inLarger Tube
2.4.2Grow Cells to
25 mLOvernight inLarger Tube
2.4.4Transfer Cells To 0.5 or 1.0L
2.4.4Transfer Cells To 0.5 or 1.0L
Inputs
Outputs
2.4.3RecordOpticalDensity
Measurements
2.4.3RecordOpticalDensity
Measurements
2.5 Induce Cells Level 2 – Process Map
2.5.1Verify that
Optical DensityHas reached
1.0
2.5.1Verify that
Optical DensityHas reached
1.0
2.5.2Induce
Cells
2.5.2Induce
Cells
2.5.3Grow Cells
Overnight at17C
2.5.3Grow Cells
Overnight at17C
Inputs
Outputs
2.6Level 2 – Process Map
2.6.1Record Optical
Density
2.6.1Record Optical
Density
2.6.2Take
Aliquots
2.6.2Take
Aliquots
2.6.4FreezePellets
2.6.4FreezePellets
Inputs
Outputs
2.6.3Centrifuge
Tubes
2.6.3Centrifuge
Tubes
2.6.5Manually Update
FermentationStorage DatabaseWith OD
Measurements
2.6.5Manually Update
FermentationStorage DatabaseWith OD
Measurements
2.7 Conduct Fermentation AnalysisLevel 2 – Process Map
2.7.1Add
BuffersTo
Pellets
2.7.1Add
BuffersTo
Pellets
2.7.2SonicateSamples
2.7.2SonicateSamples
2.7.3Centrifuge
Samples
2.7.3Centrifuge
Samples
Inputs
Outputs
2.7.4Perform
SDS-Page
2.7.4Perform
SDS-Page
2.7.5Take
Picture &Label Gel
Photo
2.7.5Take
Picture &Label Gel
Photo
2.7.6ConvertImage
To.jpg
2.7.6ConvertImage
To.jpg
2.7.7Upload .jpg
To Fermentation
StorageDatabase
2.7.7Upload .jpg
To Fermentation
StorageDatabase
3. Protein Purification with Ni-NTA Affinity Column – Input/Output Model
3Protein Purification
With Ni-NTAAffinity Column
3Protein Purification
With Ni-NTAAffinity Column
Inputs•Photograph with Information RegardingExpression level, Solubility and OD Data•Pellet in -20C Freezer
Outputs•Protein Samples in Microtube•Protein Data Sheet•ExPASy Calculation•Gel ElectrophoresisPhotograph with ProteinPurity, Yield, and MW•Mass Spectrometry dataUploaded to SPINE
3. Protein Purification with Ni-NTA Affinity Column Level 1 – Process Map
3.1Calculate Molecular
Weight and pI
3.1Calculate Molecular
Weight and pI
3.2Prepare
Samples forNi-NTA Affinity
Column
3.2Prepare
Samples forNi-NTA Affinity
Column
3.4Analyze
Ni-NTA ElutionFractions
3.4Analyze
Ni-NTA ElutionFractions
Inputs
Outputs
3.3Run Samples
Through Ni-NTAColumn
3.3Run Samples
Through Ni-NTAColumn
3.5PrepareProtein
Samples For Screening
3.5PrepareProtein
Samples For Screening
3.1 Calculate Molecular Weight & pILevel 2 – Process Map
3.1.1Access
ZebaView
3.1.1Access
ZebaView
3.1.2Copy &
Paste ORFSequence
Into ExPASy
3.1.2Copy &
Paste ORFSequence
Into ExPASy
3.4Run ExPASy
3.4Run ExPASy
Inputs
Outputs
3.1.3Add His Tag toORF Sequence
3.1.3Add His Tag toORF Sequence
3.2 Prepare Samples for Ni-NTA Affinity ColumnLevel 2 – Process Map
3.2.1Re-suspend
Pellets
3.2.1Re-suspend
Pellets
3.2.2SonicateSamples
3.2.2SonicateSamples
Inputs
Outputs
3.2.3CentrifugeSamples
3.2.3CentrifugeSamples
3.4 Analyze Ni-NTA Elution FractionsLevel 2 – Process Map
3.4.1Record OD
Measurements
3.4.1Record OD
Measurements
3.4.2Perform SDS-
PAGE
3.4.2Perform SDS-
PAGE
3.4.4Perform
MassSpectroscopy
3.4.4Perform
MassSpectroscopy
Inputs
Outputs
3.4.3Pool Fractions& Determine
Concentrations
3.4.3Pool Fractions& Determine
Concentrations
3.4.4 Perform Mass SpectroscopyLevel 3 – Process Map
3.4.4.1Run Spectrum
3.4.4.1Run Spectrum
3.4.4.2Copy & Paste
Into Excel
3.4.4.2Copy & Paste
Into Excel
3.4.4.4Import .gifFile intoSPINE
3.4.4.4Import .gifFile intoSPINE
Inputs
Outputs
3.4.4.3Save File as
.gif
3.4.4.3Save File as
.gif
3.5A Prepare Protein Samples for Screening – HSQC (MJ9)Level 2 – Process Map
3.5.1ASelect Buffer
Based onpI
3.5.1ASelect Buffer
Based onpI
3.5.2ACalculateAmount
Of Protein
3.5.2ACalculateAmount
Of Protein
3.5.4APrepare
Protein DataPackage
3.5.4APrepare
Protein DataPackage
Inputs
Outputs
3.5.3AExchange Buffer &
Concentrate
3.5.3AExchange Buffer &
Concentrate
3.5B Prepare Protein Samples for Screening – Analytical Gel Filtration with Static / Dynamic Light Scattering
Level 2 – Process Map
3.5.1BAdjust
ConcentrationTo < 3 mgs/ml
3.5.1BAdjust
ConcentrationTo < 3 mgs/ml
3.5.2BAdd
Reagents
3.5.2BAdd
Reagents
3.5.4BPrepare
Protein DataPackage
3.5.4BPrepare
Protein DataPackage
Inputs
Outputs
3.5.3BFreezeSample
3.5.3BFreezeSample
4. Conduct HSQC Screening – Input / Output Model
4Conduct HSQC
Screening
4Conduct HSQC
Screening
Inputs•15N Protein SamplesIn HSQC Buffer•Protein Information Sheet•ExPASy Calculation•Gel Picture•Mass Spec Data•NMR Request Form
Outputs•2D HSQC SpectrumWith Priority Score•Archived NMR data•.jpg image•SPINE / SPINS updatedWith NMR Spectra & Score•NMR Sample replaced in Microtube
4. Conduct HSQC ScreeningLevel 1 – Process Map
4.1Deposit
15N SampleInto NMR
Refrigerator
4.1Deposit
15N SampleInto NMR
Refrigerator
4.2PrepareSampleIn NMRTube
4.2PrepareSampleIn NMRTube
4.3Collect 1D
HSQC Data
4.3Collect 1D
HSQC Data
Inputs
Outputs
4.4Collect
2D HSQCData
4.4Collect
2D HSQCData
4.5Rate Data
Qualtiy
4.5Rate Data
Qualtiy
4.6Store HSQCData
4.6Store HSQCData
4.7Update
SPINS/SPINE withNMR
Spectrum
4.7Update
SPINS/SPINE withNMR
Spectrum
4.1 Deposit 15N NMR Samples into NMR RefrigeratorLevel 2 – Process Map
4.1.1Contact NMR
SpectroscopistFor NMR
Availability
4.1.1Contact NMR
SpectroscopistFor NMR
Availability
4.1.2Bring 15N NMR
Samples toNMR Lab
4.1.2Bring 15N NMR
Samples toNMR Lab
4.1.4Place ProteinData Package
On NMR Spectroscopists
Desk
4.1.4Place ProteinData Package
On NMR Spectroscopists
Desk
Inputs
Outputs
4.1.3Place NMR
Samples intoNMR
Refrigerator
4.1.3Place NMR
Samples intoNMR
Refrigerator
4.2 Prepare Sample in NMR TubeLevel 2 – Process Map
4.2.1Clean & DryNMR Tube
4.2.1Clean & DryNMR Tube
4.2.2Add 5% D2)(if needed)
4.2.2Add 5% D2)(if needed)
4.2.4Load NMR Sample into
NMR for Screening
4.2.4Load NMR Sample into
NMR for Screening
Inputs
Outputs
4.2.3Place SampleIn Clean NMR
Tube
4.2.3Place SampleIn Clean NMR
Tube
4.3 Collect 1D HSQC DataLevel 2 – Process Map
4.3.1Conduct initial
ManualShimming
4.3.1Conduct initial
ManualShimming
4.3.2Upload
StandardPulse sequence
HSQC
4.3.2Upload
StandardPulse sequence
HSQC
4.3.4Process 1D
HSQC
4.3.4Process 1D
HSQC
Inputs
Outputs
4.3.3Record 1D
Spectra
4.3.3Record 1D
Spectra
4.4 Collect 2D HSQC DataLevel 2 – Process Map
4.3.1Conduct initial
ManualShimming
4.3.1Conduct initial
ManualShimming
4.3.4Process 2D
HSQC
4.3.4Process 2D
HSQC
Inputs
Outputs
4.3.3Record 1D
Spectra
4.3.3Record 1D
Spectra
4.5 Rate Data QualityLevel 2 – Process Map
4.5.1Display
Processed2D
Spectra
4.5.1Display
Processed2D
Spectra
Inputs
Outputs
4.5.2Select Score
FromEstablished Categories
4.5.2Select Score
FromEstablished Categories
4.6 Store HSQC DataLevel 2 – Process Map
4.6.1Save NMRSpectrum
With Score
4.6.1Save NMRSpectrum
With Score
Inputs
Outputs
4.6.2Email
BiochemistData Directory
Location
4.6.2Email
BiochemistData Directory
Location
4.7 Update SPINE/SPINS with NMR SpectrumLevel 2 – Process Map
4.7.1Use SPINSInterface
to upload HSQC to
SPINS/SPNE
4.7.1Use SPINSInterface
to upload HSQC to
SPINS/SPNE
Inputs
Outputs
5. Analytical Gel Filtration with Static/Dynamic Light Scattering – Input/Output Model
5Analytical Gel Filtration with Static/Dynamic
Light Scattering
5Analytical Gel Filtration with Static/Dynamic
Light Scattering
Inputs•Concentrated ProteinSample with DTT, Arg,and Glycerol Frozen at -80C•Protein Info Sheet,ExPASy Calc, GelPicture and Mass SpecData
Outputs•Results uploaded to SPINE
5. Analytical Gel Filtration with Static/Dynamic Light ScatteringLevel 1 – Process Map
Inputs
Outputs
6. Preparative Gel Filtration Under Monodisperse Conditions – Input/Output Model
6Preparative
Gel FiltrationUnder
MonodisperseConditions
6Preparative
Gel FiltrationUnder
MonodisperseConditions
Inputs•MonodisperseSample ConditionsEntered into SPINE
Outputs•Protein Samples in Monodisperse BufferCondition•Gel Electrophoresis Picture of Protein PurityAnd Yield
6. Conduct Preparative Gel Filtration Under Monodisperse ConditionsLevel 1 – Process Map
6.1Review
MonodisperseBuffer
Conditions
6.1Review
MonodisperseBuffer
Conditions
6.2Thaw Ni-NTAPurifiedSample
6.2Thaw Ni-NTAPurifiedSample
6.3Perform
FPLCOn Ni-NTA
PurifiedSample
6.3Perform
FPLCOn Ni-NTA
PurifiedSample
Inputs
Outputs
6.4Analyze
FPLCResults
6.4Analyze
FPLCResults
6.5Adjust Protein
Conc. To10 mgs/ml& Freeze
6.5Adjust Protein
Conc. To10 mgs/ml& Freeze
6.6Prepare Protein
DataPackage
6.6Prepare Protein
DataPackage
6.4 Analyze FPLC ResultsLevel 2 – Process Map
6.4.1Record OD
Results
6.4.1Record OD
Results
6.4.2Perform
SDS-PAGE
6.4.2Perform
SDS-PAGE
6.4.3Pool
Fractions&
DetermineConc.
6.4.3Pool
Fractions&
DetermineConc.
Inputs
Outputs
6.4.4Perform
MassSpec.
6.4.4Perform
MassSpec.
6.4.5Perform
SDS-PAGEOn PooledFractions
6.4.5Perform
SDS-PAGEOn PooledFractions
7. Deliver Samples for Crystallographic Screening – Input/Output Model
7Deliver Protein
Samples forCrystallographic
Screening
7Deliver Protein
Samples forCrystallographic
Screening
Inputs•Concentrated,Frozen,Protein Sample•Protein InformationSheet•Gel Picture with Protein Purity and YieldInformation•Mass Spec. Data
Outputs•Concentrated ProteinSample•Federal Express TrackingNumber•Federal Express SamplePickup
7. Deliver Protein Samples for Crystallographic ScreeningLevel 1 – Process Map
7.1Concentrate
& FreezeProtein Sample
(10 mgs/ml)
7.1Concentrate
& FreezeProtein Sample
(10 mgs/ml)
7.2Arrange
For FederalExpressPickup
7.2Arrange
For FederalExpressPickup
7.3PrepareFederalExpressSamplePackage
7.3PrepareFederalExpressSamplePackage
Inputs
Outputs
7.4Deliver
Package toFederalExpress
Site
7.4Deliver
Package toFederalExpress
Site
7.5ContactCrystall-ographer
7.5ContactCrystall-ographer
7.6Await
ScreeningResults
7.6Await
ScreeningResults
8. Manufacture Enriched Protein for Structural Studies – Input/Output Model
8Manufacture
Enriched ProteinFor Structural
Studies
8Manufacture
Enriched ProteinFor Structural
Studies
Inputs•HSQC ScreeningResults•CrystallographicScreening Results•SPINE Updated withScreening Results
Outputs•1mM 13C,15N EnrichedProtein Samples in HSQCBuffer in Microtube or•10 mgs/ml SeMet Labeled Protein in Microtube•Protein Data Package•13C, SeMet Ordered
8. Manufacture Enriched Protein for Structural StudiesLevel 1 – Process Map
8.1DiscussProjectPriority
8.1DiscussProjectPriority
8.2Conduct
Small-ScaleExpression
8.2Conduct
Small-ScaleExpression
8.3PerformProtein
Expression
8.3PerformProtein
Expression
Inputs
Outputs
8.4Execute Protein
PurificationWith Ni-NTA
Affinitycolumn
8.4Execute Protein
PurificationWith Ni-NTA
Affinitycolumn
8.5Conduct
PreparativeGel
Filtration
8.5Conduct
PreparativeGel
Filtration
8.6Run 2DHSQCNMR
Spectra
8.6Run 2DHSQCNMR
Spectra
13C, 15N
8.1 Discuss Project Priority at Project MeetingLevel 2 – Process Map
8.1.1Check
MolecularWeight
OfCandidate
8.1.1Check
MolecularWeight
OfCandidate
8.1.2Review
ScreeningResults
8.1.2Review
ScreeningResults
8.1.3Check
ZebaviewCompetition
Analysis
8.1.3Check
ZebaviewCompetition
Analysis
Inputs
Outputs
8.1.4Assess
CollaboratorAvailability
8.1.4Assess
CollaboratorAvailability
8.1.5Order
Reagents
8.1.5Order
Reagents
8.2 Conduct Small-Scale ExpressionLevel 2 – Process Map
8.2.1Pick Cone used
For InitialExpression
8.2.1Pick Cone used
For InitialExpression
Inputs
Outputs
8.2.2Repeat1.4.9-1.5.9
8.2.2Repeat1.4.9-1.5.9
8.3 Perform Protein ExpressionLevel 2 – Process Map
8.3.1Repeat2.2-2.7
8.3.1Repeat2.2-2.7
Inputs
Outputs
8.4 Execute Protein Purification with Ni-NTA ColumnLevel 2 – Process Map
8.4.1Repeat Steps
3.2.1 –3.4.3
8.4.1Repeat Steps
3.2.1 –3.4.3
Inputs
Outputs
8.5A Conduct Preparative Gel Filtration – 13C, 15NLevel 2 – Process Map
8.5.1ARun FPLC in
Low-SaltBuffer
8.5.1ARun FPLC in
Low-SaltBuffer
8.5.3ARepeat3.5.2A-3.5.4A
8.5.3ARepeat3.5.2A-3.5.4A
Inputs
Outputs
8.5.2ARepeat6.4.1-6.4.5
8.5.2ARepeat6.4.1-6.4.5
8.5B Conduct Preparative Gel Filtration - SeMetLevel 2 – Process Map
8.5.1BRun FPLC
In RecommendedMonodisperse
Buffer
8.5.1BRun FPLC
In RecommendedMonodisperse
Buffer
8.5.2BRepeat 6.4.1-6.4.5
8.5.2BRepeat 6.4.1-6.4.5
8.5.4BPrepare Protein
DataPackage
8.5.4BPrepare Protein
DataPackage
Inputs
Outputs
8.5.3BAdjust Protein
Conc. To10 mgs/ml
8.5.3BAdjust Protein
Conc. To10 mgs/ml
8.6 2D HSQC NMR Spectrum of 13C,15NLevel 2 – Process Map
8.6.1Repeat
4.1, 4.2.1-4.2.4
8.6.1Repeat
4.1, 4.2.1-4.2.4
8.6.2Load
Previous2D HSQC
Parameters
8.6.2Load
Previous2D HSQC
Parameters
8.6.3Repeat4.4.1-4.4.3
8.6.3Repeat4.4.1-4.4.3
Inputs
Outputs
8.6.4Overlay13C,15N2D HSQC
w/ previous2D HSQC
8.6.4Overlay13C,15N2D HSQC
w/ previous2D HSQC
8.6.5VerifySpecta
Are Identical
8.6.5VerifySpecta
Are Identical
9. Deliver Enriched Protein Samples for Structure Determination– Input/Output Model
9Deliver EnrichedProtein Samples
For StructureDetermination
9Deliver EnrichedProtein Samples
For StructureDetermination
Inputs• 1mM 13C,15N EnrichedProtein SamplesIn HSQC BufferIn Microtube or• 10 mgs/ml SeMetLabeled Protein in Microtube• Protein Data Package
Outputs•Federal Express Package Pickup•Federal Express Tracking Number
9. Deliver Enriched Protein Sample for Structure DeterminationLevel 1 – Process Map
9.1Repeat
7.2-7.4
9.1Repeat
7.2-7.4
9.3Await FurtherInstructions
FromColloborator
9.3Await FurtherInstructions
FromColloborator
Inputs
Outputs
9.2Contact
CollaboratorWith FedExInformation
9.2Contact
CollaboratorWith FedExInformation
