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Comparative Physiology @ Guelph

TnT Purification - 2008

  • Rat cTnT in Pet24a(+) plasmid 
  • Plasmid is resistant to Kanamycin
  • working concentration  of Kanamycin is 60 ug/ml 
  • To start growth create starter culture: at 5 PM innoculate 2 x 50 ml volume of LB (with Kanamycin) with freshly transformed bacteria.
  • grow overnight at 37 oC, 200 rpm
  • Put the 6 X 1 litres of TY in shaker so they are ready next morning
  • early next morning add 10 mls of LB starter culture to each litre of TY (with kanamycin)
  •  grow at 37 oC 250 rpm until OD600 = 0.6-0.8
  • Innoculate each litre with IPTG working concentration = 0.4 mM
  • up a 6 Liter pellet,
  • collect pellet into single bottle 
  • Freeze overnight
  • Bring volume up to 250ml with 50mM Tris pH 7.4, 0.1% TritonX100, 1mM PMSF
  • Mix well, you may need to use a Polytron if it is very thick 
  • Transfer to a glass beaker and while stirring add drop by drop 
  • 25mg freshly made lysozyme (have it at 10mg/ml in water = 2.5ml solution) 
  • Keep stirring until it gets sticky, then continue stirring for 3-4 hours at room temp or overnight in the cold room at 4° 
  • Sonicate on #10 setting in multiple 2 minute bursts on ice until it gets less sticky 
  • Centrifuge at 14,000 for 20-30 minutes, save super and discard pellet 
  • Measure volume of super and add 16.4g/100ml Ammonium Sulfate (30%) 
  • Stir to dissolve and check pH, do not let pH drop below 7.0 (pH 7.5 is ideal) 
  • Centrifuge at 14,000 for 20 minutes and save super and discard pellet 
  • Measure volume of super and add 12.7g/100ml Ammonium Sulfate 
  • Centrifuge at 14,000 for 20 minutes, save pellet and discard super this time 
  • Dissolve pellet in 25-30ml DE buffer
  • Dialyze this in 1 Liter DE buffer, changing 2-3 times



need enough antibiotic for 6200 ml of buffer

= 6000 TY for expression

– 100 LB for starter culture

– 100 LB for agar plates

working concentration of Kanamycin is 60 ug/ml

 total: 0.372 g

disolve antibiotic (salt) in 6.2 ml water

filter sterilize through syringe filter.

Store frozen in tube wrapped in tinfoil.

add 1 ml/litre of media



working concentration = 0.4 mM

MW = 238.3

need 0.95 g/litre of media

for 6 litres need 0.571 g

disolve in 6 ml of water and filter steralize

add 1 ml to each litre

DE Column Buffer

25 mM Tris

6 M urea


15 mM BME

pH 8.0 


 Elute using 0-250 mM NaCl (in DE Buffer). Eluted at around 0.2 M NaCl


Dialyze peak fractions into CM buffer



CM Buffer

  6M Urea

      25mM MES

      1mM EGTA

      15mM BME

 pH 6


Elute using 0-0.3 M NaCl (in CM Buffer). Eluted at around 0.2 M NaCl.