Koeris/Notebook/2007-1-24: Difference between revisions
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==Sample preparation== | ==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. | 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=== | ===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. | 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. | ||
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|- | |- | ||
|} | |} | ||
'''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 | |||
{| border="1" | |||
! 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 | |||
{| border="1" | |||
! 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 | |||
{| border="1" | |||
! 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== | ==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). | 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). |
Revision as of 22:16, 24 January 2007
=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).