Workflow of SeMet Protein Preparation Yingyi Fang Haleema Janjua.
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Transcript of Workflow of SeMet Protein Preparation Yingyi Fang Haleema Janjua.
Workflow of SeMetProtein Preparation
Yingyi Fang
Haleema Janjua
WorkflowDay 1:
Two Step Purification
Using AKTAxpress
Day 2:
•SDS-Page
•Maintain AKTA system
•Continuation of purification
Day 3:
Sample prep:
•SDS-PAGE analysis
•Pool fractions
•Concentrate
•Aliquot
Day 4:
•Final SDS-Page
•Mass Spec
•Analyze aggregation screening
Day 5:
•Bulk upload
•Analyze aggregation
screening results and upload
Day One
1Equilibrate AKTAxpress
1Equilibrate AKTAxpress
2Obtain necessary info
for each protein
2Obtain necessary info
for each protein
33
5Sonicate cell suspension
(Total)
5Sonicate cell suspension
(Total)
7Filter supernatant (0.45m)
7Filter supernatant (0.45m)
8Load sample onto
AKTAxpress and runovernight
8Load sample onto
AKTAxpress and runovernight
4Resuspend pellet in
Binding Buffer
4Resuspend pellet in
Binding Buffer
Retrieve pellet from freezer
6Centrifugation
(Soluble)
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 AKTAxpress: 1) Wash Sample loop 2) Recharge Nickel column
Day 3
Pool fractions Based Unicorn
Result and SDS-PAGE
Pool fractions Based Unicorn
Result and SDS-PAGE
Concentrate Sample to ~10mg/ml
By AmiconUltrafree
Device(MWCO 5K)
Concentrate Sample to ~10mg/ml
By AmiconUltrafree
Device(MWCO 5K)
Determine Concentration at 280nm by Diluting protein with 6M Guanidine+ 10mM Tris, pH 7.5
-Aliquot proteins in the following manner:1. 0.45ml in 1.7ml Eppendorf tube for HWI 2. 50µl in vial for Aggregation Screening 3. ~2ml in PCR strips (50µl/tube) for
Columbia* 4. Store samples above and leftover
using LN2
5. 20µl in Eppendorf tube for SDS-PAGE and Mass spec (4C)
*In case of volume less than 1ml request more fermentation
In case of precipitation• Stop further concentrating• Remove precipitate by
centrifugation• Analyze supernatant
Day Four
Final SDS-PAGE For Purity
Mass Spec To
Confirm MW
Aggregation screening files analyzed
Proteins with MWgreater than or less
than 500 Daltons from MW reported in
Expression ID are held and submitted for
LC-MS analysis (PeterLobel’s group). DNASequencing archive
checked to verify sequence when needed.
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.
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 (>∆500)
-Check DNA sequence results-If not correct send for LC/MS/MS
analysis
Data Management
Purification Record
Purification Upload
Purification Batch Upload
Data from the purification upload is pasted here
Purification Batch Upload
Purification Batch Upload
PST ID from SPiNE is pasted into PST upload.
The volume, concentration and location is inputted into the spreadsheet.
Data from PST upload is pasted here
PST Upload
PST Upload
PST Upload
The first 2 columns of Purification upload is pasted here
Batch Images Upload
Batch Images Upload
Batch Images Upload
Aggregation Screening
Aggregation Screening
Aggregation Screening
Establish the peaks of the light scattering to determine the recovered mass, peak mass and the molecular weight
Aggregation Screening
Aggregation Screening
Aggregation Screening
Aggregation Screening
Contents from the As Upload worksheet is pasted here
Aggregation Screening
Aggregation Screening
Aggregation Screening