Koeris/Notebook/2007-1-24
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__TOC__ | __TOC__ | ||
| + | =Expression of [His]<sub>6</sub> 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. | ||
| + | {| border="1" | ||
| + | ! Reagent !! 1ul load !! 2ul load !! 3ul load !! 5ul load | ||
| + | |- | ||
| + | ! Sample !! 1ul !! 2ul !! 3ul !! 5ul | ||
| + | |- | ||
| + | ! SDS laoding buffer 2X !! 5ul !! 5ul !! 5ul !! 5ul | ||
| + | |- | ||
| + | ! ddH<sub>2</sub>O !! 4ul !! 3ul !! 2ul !! 0ul | ||
| + | |- | ||
| + | |} | ||
| + | ==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). | ||
Revision as of 17:44, 24 January 2007
Contents |
=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 |
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).
