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
(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
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.
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.