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Workflow of the Manual Purification of N/NC5-enriched proteins. Karishma G. Shetty October 7 th , 2005. Criteria for manual purification. If the extinction coefficient is less than 4000. If the protein had previously failed purification in the AKTA Express. Purification Workflow. Day One - PowerPoint PPT Presentation
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1
Workflow of the Manual Purification of N/NC5-enriched proteins
Karishma G. ShettyOctober 7th, 2005
2
Criteria for manual purification
• If the extinction coefficient is less than 4000.
• If the protein had previously failed purification in the AKTA Express
3
Day One
Two Step Purification
Day Two
SDS-PAGE of fractions
collected by AKTA
Day Three
Pool desired fractionsand
Concentrate
Day Five
Final SDS-PAGEand
Mass Spec
Day Six
Bulk Uploadand
NMR Sample Prep
Day Four
Buffer Exchangeand
Concentrate
Outline
4
1Obtain necessary info from
Spine for each protein
1Obtain necessary info from
Spine for each protein
4Sonicate cell suspension
(Total)
4Sonicate cell suspension
(Total)
Day One
6Filter supernatant (0.45m)
(Soluble)
6Filter supernatant (0.45m)
(Soluble)3
Resuspend pellet in 25 ml of Lysis Buffer
3Resuspend pellet in 25 ml of Lysis Buffer
2Get pellet from
freezer
5Obtain supernatant by
centrifugation
7Prepare Ni-NTA
Superflow column
8Pour the supernatant into
The Ni-NTA Superflow Column (F.T.)
9Wash the Column with
50ml of Wash Buffer(W)
9Wash the Column with
50ml of Wash Buffer(W)
10Elute the protein from the
Ni-NTA column using10ml of Elution Buffer (E)
10Elute the protein from the
Ni-NTA column using10ml of Elution Buffer (E)
11Run an SDS PAGE Gel Of the Total, Soluble,
Flow through, Wash andElution Samples
11Run an SDS PAGE Gel Of the Total, Soluble,
Flow through, Wash andElution Samples
12Inject the Samples into
the AKTA Express and let them run overnight .
12Inject the Samples into
the AKTA Express and let them run overnight .
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SDS Page
6
Day Two
Analyze chromatographand decide which fractions to run
for SDS-PAGE
Decide which fractions to pool based on result of chromatograph and SDS-PAGE
MAINTAIN THE AKTA
7
Day Three
Pool fractions Based Unicorn
Result
Pool fractions Based Unicorn
Result
Concentrate Sample to 0.5-1.0 mMBy AmiconUltrafree
Device
Concentrate Sample to 0.5-1.0 mMBy AmiconUltrafree
Device
Determine Concentration at 280nm by diluting protein with 6M Guanadine + 10mM Tris, pH 7.5
In case of precipitation
Stop further concentrating,remove precipitate bycentrifugation andAnalyze supernatant
8
Day Four
Buffer exchange into selected NMR Buffers using desalting
columns if there is enough protein.
Concentrate Sample to 0.5-1.0 mM by Amicon Ultrafree Device
Concentrate Sample to 0.5-1.0 mM by Amicon Ultrafree Device
Upload purification record to SPiNE and obtain PST IDs
Bulk Purification Uploads into SPiNE – Purification Template
SR361.002
9
Day Five
Final SDS-PAGE
GelFor Purity
Mass Spec To
Confirm MW
Proteins with MW with adeviation greater than 500 Daltons from the reported value in SPiNE are held and submitted for LC-MS analysis (PeterLobel’s group).
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Day Six
SDS-PAGEpictures
taken
InformationPlaced in SPINsIn JPEG format
InformationUploaded ontoSPiNE
NMR Samples Made, placed in the inbox and email sent out (Swapna, Tom and Rong)
Protein information uploaded into SPINS from SPiNE.
Upload PSTInformationinto SPiNE
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Proteins attemptedto be purified
147 ProteinsSuccessfully purified
Purification Flowchart
Failed purification
42 Proteins (28.6%) 105 Proteins (71.4%)
E x S <= 4 E x S > 4
10 Proteins (23.8%)32 Proteins (76.2%)
No Binding or Aggregates in GF
Change the buffer & purifyManually or make a new Construct.NOTE: Test for hex-his tag.
12
Proteins attemptedto be purified
147 ProteinsSuccessfully purified
Purification Flowchart
Failed purification
42 Proteins (28.6%) 105 Proteins (71.4%)
E x S <= 8 E x S > 8
7 Proteins (16.67%)35 Proteins (83.33%)
Change the buffer & purifyManually or make a new Construct.NOTE: Test for hex-his tag.
No Binding or Aggregates in GF