1 / 31

Workflow of SeMet Protein Preparation

Workflow of SeMet Protein Preparation. Yingyi Fang Haleema Janjua. 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:

makaio
Download Presentation

Workflow of SeMet Protein Preparation

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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. 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

  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 Lobel’s 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 (>∆500) -Check DNA sequence results -If not correct send for LC/MS/MS analysis

  9. Data Management

  10. Purification Record

  11. Purification Upload

  12. Purification Batch Upload Data from the purification upload is pasted here

  13. Purification Batch Upload

  14. Purification Batch Upload

  15. PST ID from SPiNE is pasted into PST upload. The volume, concentration and location is inputted into the spreadsheet.

  16. PST Upload Data from PST upload is pasted here

  17. PST Upload

  18. PST Upload

  19. Batch Images Upload The first 2 columns of Purification upload is pasted here

  20. Batch Images Upload

  21. Batch Images Upload

  22. Aggregation Screening

  23. Aggregation Screening

  24. Aggregation Screening Establish the peaks of the light scattering to determine the recovered mass, peak mass and the molecular weight

  25. Aggregation Screening

  26. Aggregation Screening

  27. Aggregation Screening

  28. Aggregation Screening Contents from the As Upload worksheet is pasted here

  29. Aggregation Screening

  30. Aggregation Screening

  31. Aggregation Screening

More Related