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.

Sample setup

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.

Reagent	1ul load	2ul load	3ul load	5ul load
Sample	1ul	2ul	3ul	5ul
SDS laoding buffer 2X	5ul	5ul	5ul	5ul
ddH2O	4ul	3ul	2ul	0ul
NuPAGE reducing agent	1ul	1ul	1ul	1ul

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.

Preparation of whole cell. and soluble protein 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

1x: vol. per one MP well	Volume
lysozym 10 mg/ml	5ul
1% Brij58	12.5 ul
Lysis Buffer	7.5ul

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

1x: vol. per one MP well	Volume
1 M MgCl2	0.3ul
Benzonase gradeII 25 U/µl Merck	0.1ul
50 mM Tris-HCl pH 8.0	24.6ul

• vortex briefly, incubate at room temperature 30 min

Whole cellular extract

• put 15 µl of

1x: vol. per one MP well	Volume
4x SDS loading buffer	7.5ul
1 M DTT	2.22ul
H2O	5.25ul

• 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).