Workflow of SeMet Protein Preparation

1
Workflow of SeMetProtein Preparation

• Yingyi Fang
• Haleema Janjua

2
Workflow

• Day 2
• SDS-Page
• Maintain AKTA system
• Continuation of purification

Day 1 Two Step Purification Using AKTAxpress

• Day 5
• Bulk upload
• Analyze aggregation screening results and upload

• Day 3
• Sample prep
• SDS-PAGE analysis
• Pool fractions
• Concentrate
• Aliquot

• Day 4
• Final SDS-Page
• Mass Spec
• Analyze aggregation screening

3
Day One
1 Equilibrate AKTAxpress
8 Load sample onto AKTAxpress and run overnight
2 Obtain necessary info for each protein
7 Filter supernatant (0.45mm)
3
6 Centrifugation (Soluble)
Retrieve pellet from freezer
4 Resuspend pellet in Binding Buffer
5 Sonicate cell suspension (Total)
4
Analyze chromatographand decide which fractions
to run for SDS-PAGE
Day Two
Decide which fractions to pool based on result of
chromatograph and SDS-PAGE
Maintain AKTAxpress 1) Wash Sample loop 2)
Recharge Nickel column
5
Day 3
Pool fractions Based Unicorn Result and
SDS- PAGE
Concentrate Sample to 10mg/ml By
Amicon Ultrafree Device (MWCO 5K)
Determine Concentration at 280nm by Diluting
protein with 6M Guanidine 10mM Tris, pH 7.5

• -Aliquot proteins in the following manner
• 0.45ml in 1.7ml Eppendorf tube for HWI
• 50µl in vial for Aggregation Screening
• 2ml in PCR strips (50µl/tube) for Columbia
• Store samples above and leftover using LN2
• 20µl in Eppendorf tube for SDS-PAGE and Mass spec
  (4oC)
• In case of volume less than 1ml request more
  fermentation

• In case of precipitation
• Stop further concentrating
• Remove precipitate by centrifugation
• Analyze supernatant

6
Day Four
Mass Spec To Confirm MW
Final SDS-PAGE For Purity
Proteins with MW greater than or less than 500
Daltons from MW reported in Expression ID are
held and submitted for LC-MS analysis
(Peter Lobels group). DNA Sequencing
archive checked to verify sequence when needed.
Aggregation screening files analyzed
7
Day Five
SDS-PAGE pictures are taken using AlphaImager,
labeled by Adobe and saved as JPEG into
individual folder on Spins server
Chromatograph and Mass Spec images are saved as
JPEG into individual folder on Spins server
Excel notebook file is completed with entire
record of process.
Images are uploaded onto SPiNE. Comments about
protein are listed.
8
Recommended Recovery
Failed Purification -make a new construct and
purify again -referment Precipitation upon
concentrating -Optimize buffer condition Low
yield -Multiple liter fermentation needed Low
purity -Additional Ion exchange
chromatography Molecular weight out of range
(gt?500) -Check DNA sequence results -If not
correct send for LC/MS/MS analysis
9
Data Management
10
Purification Record
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Purification Upload
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Purification Batch Upload
Data from the purification upload is pasted here
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Purification Batch Upload
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Purification Batch Upload
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PST ID from SPiNE is pasted into PST upload.
The volume, concentration and location is
inputted into the spreadsheet.
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PST Upload
Data from PST upload is pasted here
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PST Upload
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PST Upload
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Batch Images Upload
The first 2 columns of Purification upload is
pasted here
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Batch Images Upload
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Batch Images Upload
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Aggregation Screening
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Aggregation Screening
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Aggregation Screening
Establish the peaks of the light scattering to
determine the recovered mass, peak mass and the
molecular weight
25
Aggregation Screening
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Aggregation Screening
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Aggregation Screening
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Aggregation Screening
Contents from the As Upload worksheet is pasted
here
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Aggregation Screening
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Aggregation Screening
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Aggregation Screening