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