1. Protein Sample Preparation For NMR Screening
Chioma Nwosu
Mei Jiang
Rong Xiao
February 16th, 2007

2. Purification Workflow
Day One
Two Step(Ni, Gel filtration) Purification using AKTAxpress
Day Two
Continuation of purification
SDS-PAGE of fractions
collected by AKTA
Day Three
Pool desired fractions.
Estimate sample numbers and concentrate.
Start buffer exchange .
Purification record upload.
Day Six
Bulk Upload.
NMR Sample delivery.
Outline
Day Five
Measure OD280 to determine concentration.
Final SDS-PAGE and Mass Spec.
Day Four
Continue buffer exchange and concentrating.

3. Day One 1 Obtain necessary info from Spine for each protein 7
Inject the Samples into
the AKTA Express and let
them run overnight . 6
Filter supernatant (0.45mm)
(Soluble) 5
Obtain supernatant by
centrifugation 2
Get pellet from freezer 3
Resuspend pellet in
25 ml of Binding Buffer 4
Sonicate cell suspension
(Total)

4. Analyze chromatograph and decide which fractions to run for SDS-PAGE
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 the AKTA:
Wash samples loops
Recharge Ni columns.

5. Day Three In case of precipitation
Pool fractions based on SDS PAGE and Unicorn results. Determine how many buffer exchanges could be done.
Start concentrating.
If enough for
3 samples: 9 ml samples: 6 ml
1 sample: 0.5 ml
In case of precipitation
Stop further concentrating,
remove precipitate by
centrifugation and
Analyze supernatant
Prepare for buffer exchange by equilibrating system with
desired buffers.
Upload purification record and
request for PST IDs.

6. Mannually inject proteins by:
Day Four
Continue Buffer Exchange: pool fractions and concentrate.
Mannually inject proteins by:
Instant Run  Buffer Exchange  Run HP Trap  Choose sample numbers  Type in all information requested  Run  Follow all instructions in Dialogue Box

7. Day Five Proteins with MW with a deviation greater than
500 Daltons from the
reported value in SPiNE
are reported in SPiNE with
detailed comments.
Final
SDS-PAGE
Gel
for purity
Mass Spec To
confirm MW

8. Day Six SDS-PAGE,Mass Spec images taken NMR Samples
Made, placed in the
inbox and
sent out
(Swapna, Tom and
Rong,Mike, Ken)
SDS-PAGE,Mass Spec images taken
Images
Placed in SPINs
Protein information
uploaded into
SPINS from SPiNE.
Images
Uploaded onto
SPiNE
Upload PST
Information
into SPiNE