Patricia L. Clark
last updated: 4/21/99
Specific for labelling nascent tailspike polypeptide chains (and, as a control, Salmonella proteins)
Materials needed
biological:
Salmonella 7136 fresh
O/N in minimal media
Hi-titer (Ž1x1011 pfu) phage stock (5-/13-, or N110 for another control)
minimal media
casamino acids (10%)
Buffer R + OG [ribosome buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 150 mM KCl)
plus 80 mM octyl-gluco-pyranoside]
radioactive:
2ml of 14C-labeled L-amino
acid mixture (per phage used)
radioactive hood clear for work
geiger counter
hot pipetmen
radiation badge, safety glasses, lab coat
other:
SS34 tubes (4) and rotor
liq. N2 dewer
ice tub
Detailed protocol
1. Start (2) 50 ml cultures of 7136, using 1 ml of O/N culture, in 125 ml baffle
flasks, at 30degC.
2. Grow until cell density reaches 2x108 cells/ml. For steps 3-10, the cultures
will be treated differently:
labeling cellular stuff (R)
3. Pulse with 1 ml 14C-labeled aa (100 mCi). Wait 15 min.
4. Culture 10 min.
5. Chase with 10 ml ice cold casamino acids
6. Infect at MOI=10 (vol=60 ml)
7. Culture 90 min.
labeling nascent tailspike
(T)
6. Infect at MOI=10 (vol=50 ml).
7. Culture 75 min.
8. Pulse 1 ml 14C-aa (100 mCi).
9. Culture 10 min.
10. Chase with 10 ml
cold cas-aa.
11. Chill both cultures on ice.
12. Pour into SS34 tubes (two per culture) and spin at 11K rpm for 5 min.
13. Drain pellets and resuspend in 0.25 ml Buffer R + OG.
14. Transfer suspended cells to microfuge tubes, and freeze in liquid N2. Store
at -30degC.