Workflow of SeMet Protein Preparation Yingyi Fang Haleema Janjua

Workflow Day 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 1 Equilibrate AKTAxpress 2 Obtain necessary info for each protein 3 5 Sonicate cell suspension (Total) 7 Filter supernatant (0.45  m) 8 Load sample onto AKTAxpress and run overnight 4 Resuspend pellet in Binding Buffer Retrieve pellet from freezer 6 Centrifugation (Soluble)

Day Two Analyze chromatograph and 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 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: ml 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 LN µl in Eppendorf tube for SDS-PAGE and Mass spec (4  C) *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 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.

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

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

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

Aggregation Screening

Establish the peaks of the light scattering to determine the recovered mass, peak mass and the molecular weight

Aggregation Screening

Contents from the As Upload worksheet is pasted here

Aggregation Screening