CLONE ID: 8-27

EXPRESSION HOST: BL21(DE3)

PLASMID: pSBETa (30 mg/L Kanamycin)

pI: 10.17

USE FRESHLY TRANSFORMED CELLS!

• Mid-afternoon, 4 L of LB + 30 mg/L kanamycin, inoculated with 20 colonies each 2 L flask (fresh transformation), grown O/N.
• Pre-equilibrated CM fast Sepharose column O/N with ~700-900 mL of Column Buffer + Inhibitors pH 8 at 4°C.Column Buffer has the same composition as the Sonication Buffer (see below), except the EDTA is at 1 mM.
• Next day, take a 1 mL sample of the O/N growth. Prepare for an acrylamide gel to check expression. Cultures spun at 7K rpm 5 minutes.
• Resuspend pellets in 100 mL Sonication Buffer.
• Composition of Sonication Buffer (pH 8 at 4°C):
  • 6 M Urea
  • 50 mM Tris
  • 5 mM EDTA
  • 1% SDS

INHIBITOR (add fresh)

• • 0.4 mM PMSF
• Sonicate ~10-15x. Make sure Sonicator has been tuned recently.
• Rinse probe with MQ Water, EtOH, then MQ Water again.
• Sonicate between settings 9-10. Sonicated for ~1 minute. Stop sonicating, and let it rest for 2-3 minutes. Repeat this until the solution has the consistency of water, usually 10-15x. Use a pipet bulb to test.
• Spin 10K rpm 1 hr. Store pellet at –20°C.

 Diayze supernatent against same buffer as above but without the 1% SDS

• Following dialysis, measure conductivity and pH of protein solution, Column Buffer in, and Flow Thru Column Buffer. Avoid large changes of pH or conductivity between the buffer before/after column. Also, watch for high conductivity in the supernatant. If the conductivity is too high or too close to where it comes off, it won’t bind to the column. See page 89B.
• Load supernatant onto CM-Fast Sepharose Column
• Gradient is 800 mL x 2, 0-0.25 M NaCl.
• Run appropriate fractions on a gel.
• RcTnI comes off at conductivity of 8-14 Mhos, fractions 33-55, pg 1:89B.
• Fractions pooled, dialyzed O/N against 4 L Affigel Column Buffer (6 M Urea, 50 mM Tris-HCl, 0.2 M NaCl, 3 mM CalCl₂) + Inhibitors, pH 8 at 4°C, see pg 2:65A.
• Pre-equilibrate Affi-gel column O/N with ~600-800 mL of Column Buffer + Inhibitors pH 8 at 4°C.
• Measure conductivity and pH of supernatant, Column Buffer in, and Flow Thru Column Buffer.
• Load supernatant onto Affi-gel Column. Regnier lab run at speed 40 or less
• After loading, wash Affi-gel column with Column Buffer + Inhibits, pH 8 at 4°C.
• After washing, at the end of the day, elute with Elution Buffer (6 M Urea, 50 mM Tris-HCl, 1 M NaCl, 2 mM EDTA) + Inhibits pH 8 at 4°C. Elute O/N, ensuring you have enough tubes in the rack to last the entire night. Pump speed is 0.5 mL/min.
• Run appropriate fractions on a gel.
• RcTnI comes off at fractions ~47-59, pg 2:65B.
• Regnier lab: Re-load the second peak to get rid of pesky upper bands. Can re-run first peak to increase your yield-more work but I think that it is worth it to get more yield
• Fractions 47-59 pooled; fractions 23-46, 60-69 pooled; dialyzed O/N against 4 L MQ water + b-mercaptoethanol.Regnier lab run scan on each fraction and only save good ones to pool
• Changed water four times, at the beginning and end of each day.
• Slight white ppt. present in dialysate.
• Froze in dry ice ethanol bath.
• Lyophilized.
• Fr 47-59 yields 178 mg; Fr 23-46, 60-69 yields 20 mg. See pg 2:65B.