Protein Sample Preparation For NMR Screening

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Presentation transcript:

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

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.

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)

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.

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 2 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.

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

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

Day Six SDS-PAGE,Mass Spec images taken NMR Samples Made, placed in the inbox and email 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