Pulse/Chase Experimental Protocol

PLC 8/10/99

NOTE: This protocol is specific for labeling nascent tailspike polypeptide chains (and, as a control, Salmonella proteins)

Materials needed

biological: Salmonella 7136 fresh O/N in minimal media

Hi-titer („1x10¹¹ 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)

labeling nascent tailspike (T)

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).

Infect at MOI=10 (vol=50 ml).

7.

Culture 90 min.

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.

	labeling cellular stuff (R)	labeling nascent tailspike (T)
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).	Infect at MOI=10 (vol=50 ml).
7.	Culture 90 min.	Culture 75 min.
8.		Pulse 1 ml ¹⁴C-aa (100 mCi).
9.		Culture 10 min.
10.		Chase with 10 ml cold cas-aa.