Table of Contents
Related protocols
Materials
FPLC Buffers
- Buffer A (1L) & B (1L)
- 2L ddH2O
- Tris (MW121.4) 4.84g and adjust pH to 8.0
- Divide it into 2 parts; one part as Buffer A
- The other Part + NaCl (MW:58.44) 58.44g + EDTA (372.24) 0.372g → Buffer B
- Buffer C (1L)
- 1L H2O
- Citric Acid (MW210.14) 3.115g
- Citric Na (MW294.10) 1.523g
- EDTA (MW 372.24) 0.372g
- NaCl (MW58.44) 8.76g
Protein Expression
- Put 5 uL stock E. coli. to 5 mL of LB media, and shake it overnight at 37C, 225rpm.
- Transfer the culture above into 1L expression medium with Amp; grow @37C,225rpm unitl OD600=0.5~0.6.
- Induce by 1mM 1PTG-D2O and further shaked for 4 hours
- Centrifuge the culture @8000rpm,4C for 15min; store in deep freezer or go head for French Press
French Press
- Resuspend cells in 20mL buffer A and keeps it always in ice box; Add 50uL 2M MgCl2 (final_C=5mM); Add 250U Benzonase (Novagen); 2 small tablets Complete (Proteinase Inhibitor, Roche)
- The suspension is twice French pressed. Instruction
- Centrifuge @18000rpm,4C for 30min; get the supernanant (Keep it always in ice box)
Protein Purification
- QFF Ion-Exchange column (2 column connected together, column size:2X5mL=10mL)
- Connect the tubling of FPLC; install the 5mL sample loop; wash the column using ddH2O if it is stored in 20% Alcohol.
- Run Program #22 to wash and equilibrate the column
- Filter the supernanant by syringe filter
- Run Program #21 to apply the sample (you may need repeat several times)
- Run Program #23 to run the column; colect the proper fractions
- Gel-filtration column
- The positive fractions are pooled and pH are adjusted to 3.5 by 1M citric acid (15ml+220ul)
- Replace the 20% Alcohol in the column with Buffer C if necessary
- Concentrate it to ~4mL by centriprep (Millipore Corp)
- Apply the sample to sample loop and run Program #24 to run the column
Prepare NMR sample
- Run SDS-PAGE to check the fractions
- Prepare buffer for NMR sample
- 0.02% NaN2
- Pool the proper fractions; concentrate it using CentriPrep several times to exchange the buffer to NMR buffer
- Concentrate the sample to ~200uL, add 10% D2O