Koeris/Notebook/2007-1-24

=Expression of [His]6 constructs=

Sample preparation
Frozen samples were thawed on ice and diluted to prepare a simple Coomassie-stain gel check. Is the overexpression visible? If so it might be too much for a physiologically relevant pulldown.

Loading of samples was a big problem, they were gooey and didn't want to go into the wells. Look up sample prep protocols for bacteria.

Quick and dirty protein expression sample prep

 * 1) transfer volume X into 1.5 ml tube, X = 0.25 ml / OD
 * 2) spin 30 sec
 * 3) resuspend pellet in 30 µl H2O
 * 4) add 3 µl 1 M DTT and 11 µl 4 x SDS Loading Buffer (i.e. 10% SDS)
 * 5) 5 min at 50ºC
 * 6) 2 min at 100ºC
 * 7) spin 5 min

Preparation of whole cell extract

 * thaw 96 bacterial pellets room temperature
 * resuspend in 100 µl Lysis buffer (50 mM Tris-HCl, pH 8.0, 0.3 M NaCl, 1 mM EDTA or 0.1 mM EDTA, if NiNTA purification follows)
 * add 25 µl of


 * vortex briefly, incubate on ice 30 min
 * add 25 µl of


 * vortex briefly, incubate at room temperature 30 min

Whole cell extract

 * put 15 µl of
 * resuspend lysates, add 15 µl to the plate, mix by pipetting
 * 5 min 50°C, 2 min 100°C
 * load 7 µl on SDS-PAGE

SDS-PAGE
We're using the Invitrogen Nu-PAGE system with Novex bis-tris 4-12% gels' and MES running buffer to get the high resolution in the low kDa-range (as advertised).

NuPage sample preparation (post-extraction)
Heat samples for 2min at 85 deg C NOT at 95 deg C as we would usually do. This has to do with the composition of Invitrogen's reagents.