Comparative Physiology @ Guelph
Charles TnC Pellet Method
- Grow up an 8 Liter pellet, then centrifuge down to a single pellet in one bottle
- Bring volume up to 250ml with 10mM Tris pH 7.4 and mix well
- Centrifuge and save pellet, discard super.
- 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 4Â°
- 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 15.4g/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 22.7g/100ml Ammonium Sulfate (50%)
- Centrifuge at 14,000 for 20 minutes, save pellet and discard super this time
- Dissolve pellet in 25-30ml DE52 buffer
- Dialyze this in 1 Liter DE52 buffer, changing 2-3 times
- Run over a 40 gram DE52 column at 1ml/minute
- 6M Urea
- 25mM Tris pH 8
- 1mM EDTA
- 50mM BME or DTT
- Elution using a gradient
Need total volume of buffers to make gradient to equal 6 times column volume. If 6 times column volume = 600 ml than require 300 ml High Salt Buffer and 300 ml Running Buffer.
(High Salt Buffer High Salt Buffer (1 l)Running Buffer with 0.35 M KCl)
Add to one bottle and Running Buffer to other. These bottles are connected through a hose at their base that is closed using a clamp. Put a stir bar in Running Buffer bottle and place on stir plate. Use adjustable stage to make height of High Salt Buffer bottle equal to that of Running Buffer bottle. Intake hose going into column is placed in Running Buffer bottle and pump is turned on. Open up clamp very slightly between High Salt Buffer and Running Buffer bottles.
Allow buffer to continue moving through the column at 1 ml/min.
Start fraction collector and spec collecting 3 ml volumes. TnC eluted when buffer concentration is around 0.32 M or the bottles are about Â¾ empty.
Use gels to determine which fractions contain single band of TnC.
If unable to get fractions with single band. Utilize Phenyl sepharose column and method.
Discard Column Material when finished pour new column every time.