In vivo folding, pulse/chase protocol

CBB - last updated: February 25, 1998


(1) grow Salmonella 7136 su- culture o/n at 30degC in minimal M9 media


(2) in the morning, dilute cells ~1:40 (0.25ml culture:10 ml media) with M9 and allow to grow to a cell density of 2x10E8 cells/ml



(3) cells should be put in a 30degC H2O bath for infection

use about 0.5 ml of cell culture for general folding experiment


(4) infect with P22 phage with an m.o.i. of 10

0.5 ml of 2 x 10E8 cells/ml is ~ 1 x 10E8 cells/ml, so use 1 x 10E9 phage



(5) 1 hour after infection, transfer cells to a 24degC H2O bath

pulse the cells with 14C-labelled amino acids to a final concentration of 2mCi/ml while transferring



(6) after a 4 minute labelling period, chase with 10% acid-hydrolyzed casein by adding 100ul/ml of culture



(7) at various time points, transfer cells to lysis buffer and then immediately freeze in liquidN2

lysis buffer is made with 3X native dye with octoglucoside added



(8) for running gel, thaw samples at 4degC and load when thawed

as a control on the gel, perform one in vitro refolding experiment with wild type tspk with a time point that shows protrimer (especially), native trimer, dimer, etc.