Cameron Haase-Pettingell
updated: 6/13/01
Stock Solutions
• Tailspike: 6.6 mg/ml both phage TSPK
--7.3 mg/ml E. Coli TSPK
• 50mM Citrate pH 3
• 8 M urea/ 50 mM citrate pH 3 ( we have had problem with Pi holding pH
3)
Unfolding Tailspike --2 mg/ml TSP, 5 M urea, pH 3.0 (one hour, 20 °C)
This must be made in a low protein binding microfuge tube.
62 µL urea stock
8 µL 50 mM citrate, pH 3.0
30 µL tailspike stock
100 µl total
| Refolding Tailspike -- | 100 µg/mL TSP |
| (30 min on ice followed by refolding at 20 °C) | |
| Time points of | 0, 1, 5, 10, 15, 20, 60, 120 min. |
We make the assumption that there is not change to the protein in the denaturant.
• 2 hr sample take 10 µl into a low binding tube and initiate refolding
by adding 200 µl of refolding buffer cold (as 0f 061301 50 mM Tris pH 7.6 2
mM EDTA)
(Tris is not good for EM))
The cold refolding buffer should be added rapidly (but don't splash out
of tube) to the denatured tailspike --in the low protein binding tube-- and
the mixture pipetted up and down a few times.
• The sample was kept on ice for 30 min then shifted to 20 °C to continue
refolding.
• t=30 min 30 sec., start the 1 hr sample because it will have a 30 min
incubation on ice followed by the 1hr incubation at 20ºC.
• continueŠ
• At the end you are removing the samples at the same time staggered by
30 sec.
• 0, 1, 5, 10, 15, 20, 60, 120 min. iced add 3X gel sample buffer.