PSF
•Soluble and whole cellular protein extracts •
- Thaw 96 bacterial pellets room temperature (see Protocol 1).
- 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 | 220x | |
|---|---|---|
| Lysozyme 10 mg/ml | 5 µl | 1100 µl |
| 1% Brij58 (Sigma) | 12.5 µl | 2750 µl |
| Lysis Buffer | 7.5 µl | 1650 µl |
- Vortex briefly, incubate on ice 30 min.
- Remove an 15 µl aliquot for SDS-PAGE analyis (whole cellular extract).
- Add 25 µl of
| 1x: vol. per one MP well | 220x | |
|---|---|---|
| 1 M MgCl2 | 0.3 µl | 66 µl |
| Benzonase gradeII 25 U/µl (Merck) | 0.1 µl | 22 µl |
| 50 mM Tris-HCl pH 8.0 | 24.6 µl | 5412 µl |
- Vortex briefly, incubate at room temperature 30 min
- Centrifuge lysates 30 min 6200 rpm 4°C
- transfer 145 µl supernatant to a small-pore filter plate (Durapore MADV N 65, Millipore) to remove any remaining particles .
- Filter supernatants on a vacuum manifold into a fresh filter plate (soluble protein extract).
SDS-PAGE analysis of soluble and whole cellular extracts
- Add 15 µl of
| 1x: vol. per one MP well | 110x | |
|---|---|---|
| 4x SDS loading buffer | 7.5 µl | 825 µl |
| 1 M DTT | 2.22 µl | 244.2 µl |
| H2O | 5.25 µl | 577.5 µl |
into a 96-well PCR plate
- Add 15 µl extract to the plate, mix by pipetting
- Incubate 5 min 50°C, 2 min 100°C
- Analyse 7 µl by 15% SDS-PAGE
© 2003 by V. Sievert, Konrad Büssow last changed 12 Sep 2006