Koeris/Notebook/2007-1-24
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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
- transfer volume X into 1.5 ml tube, X = 0.25 ml / OD
- spin 30 sec
- resuspend pellet in 30 µl H2O
- add 3 µl 1 M DTT and 11 µl 4 x SDS Loading Buffer (i.e. 10% SDS)
- 5 min at 50ºC
- 2 min at 100ºC
- 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
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 cell 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).
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.
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 |