Sampling from chemostat

For low abundance, volatile modifications (e.g. crotonylation):

400 µl of culture + 100 µl of 100% TCA (20% final)

Invert tube to mix

Spin 1 min 5000 rpm

Aspirate supernatant to waste

Freeze in liquid nitrogen and store at -80C

For total protein

Collect sample volume as required (approx. 0.5 to 1 ml)

YMC is sensitive to sample volumes, especially mutants, so try limit collection vol

Spin 1 min 5000 rpm

Aspirate supernatant to waste

Freeze in liquid nitrogen and store at -80C

Lysis of pellets for western blots:

Optional: wash pellet in water to remove excess TCA (if used)

Resuspend pellet in 200 µl of 0.15 M NaOH

Vortex to resuspend

Incubate 5 min, room temp

Spin 2 min, 5000 rpm, room temp

Aspirate supernatant to waste

Resuspend pellet in 50 µl of 1X SDS buffer (see below)

Incubate 5 min at 100C

Spin 2 min, 10000 rpm, room temp

Collect supernatant to fresh tube

Load on gel (3-5 µl is plenty)

1. 2X SDS buffer recipe – make in bulk and store at RT

0.125 M Tris-HCl pH 6.8

4% SDS

30% glycerol

0.02% Bromophenol blue

2. 1X SDS buffer – make fresh for lysis

25 µl 2X SDS buffer

2 µl beta-mercaptoethanol

23 µl water