====== Related protocols ======
  * [[protocol:protpurify:spcsh3.methyl | spcSH3 sample perparation used in solid-solution comparison]]

====== 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. [[BioInstrumentsUsage|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
{{ spcsh3_qff_method.png }}

  * 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