| Dilution/Dialysis combination |
| 50mM TrisHCl, 1%(v/v) Triton X-100, 100mM NaCl, 1mM sodium EDTA, 0.1% sodium azide, 1mM DTT pH 8.0 |
| 25mM 2-(N-morpholino)ethanesulfonic acid, 8M urea, 10mM sodium EDTA, 0.1mM DTT pH 6.0 |
| 1: 100mM TrisHCl, 500mM L-arginineHCl, 2mM EDTA, 5mM reduced glutathione, 0.5mM oxidized glutathione, 0.2mM PMSF pH 8.3, 2:20mM TrisHCl, 150mM NaCl, 1mM sodium EDTA, 0.5mM DTT pH 7.5 |
| None |
| no tag |
| 8.3 |
| 4.0 |
| 2microM |
| 32h |
| GSH/GSSG/DTT/beta mercaptoethanol |
| n/a |
| 6L LB media containing 0.4% (w/v) glucose, 30mg/L chloramphenicol and 100mg/L ampicillin was inoculated with a single bacterial colony and grown until OD600 reached 0.7-1.0. 0.5mM IPTG was added and cells were grown for a further 2h before being harvested. The cell pellets were suspended in a buffer containing 50mM TrisHCl, 25% sucrose, 1mM sodium EDTA, 0.1% sodium azide and 10mM DTT pH 8.0. 1mg/ml lysozyme, 25microg/ml DNaseI and 5mM MgCl2 were added. Lysis buffer (2.5ml per ml cell suspension) containing 50mM TrisHCl, 1%(v/v) Triton X-100, 1% (sodium deoxycholate, 100mM NaCl, 0.1% sodium azide and 10mM DTT pH 8.0 was added to the cells. AFter the viscosity decreased, 10mM sodium EDTA was added and the suspension was frozen and then thawed. The cell lysate was centrifuged, and the pellets were reuspended and centrifuged in wash buffer four times. The inclusion bodies werer then suspended in 50mM TrisHCl, 1mM sodium EDTA, 0.1% sodium azide and 1mM DTT pH 8.0, centrifuged and dissolved in 10-20ml solubilization buffer and ultracentrifuged (140000g). Aliquots of the supernatant were stored at -70degC.
The protein was diluted into refolding buffer containing 5M urea to a final concentration of 2microM. This was then dialyzed for 16h at 4degC against refolding buffer containing 1-2M urea and 5mM beta-mercaptoethanol but no glutathione. The protein was then dialyzed against refolding buffer 2 for 16h. |
| Native gel band-shift assay |
| None |
| None |
| NULL |
|
|
| same protocol as for TCR-A6 alpha |