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Team:EPF Lausanne/Project/Materials Methods
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We grew E.Coli DH5a transformed with the plasmid C3 containing the [http://partsregistry.org/Part:BBa_K320001 immunotoxin] with the [http://partsregistry.org/Part:BBa_K320002 strong promoter]([http://partsregistry.org/Part:BBa_K320006 BBa_K320006] ). As a negative control we used cultures transformed with the C3 plasmid alone. | We grew E.Coli DH5a transformed with the plasmid C3 containing the [http://partsregistry.org/Part:BBa_K320001 immunotoxin] with the [http://partsregistry.org/Part:BBa_K320002 strong promoter]([http://partsregistry.org/Part:BBa_K320006 BBa_K320006] ). As a negative control we used cultures transformed with the C3 plasmid alone. | ||
A culture volume of 100 ml was spinned down. The pellets as well as the supernatents were used as samples for a protein analysis. | A culture volume of 100 ml was spinned down. The pellets as well as the supernatents were used as samples for a protein analysis. | ||
| - | The pellets were resuspended in lysis buffer containing urea and sonicated for 15 minutes. For the western blot the samples were run on an SDSpage gel and then transferred to a nitrocellulose membrane. The detection was accomplished using the following antibodies: Anti-his biotin as primary and Streptavidin-HPR as a secondary antibody. Additional to the primary antibody we applied Anti-his HRP. | + | The pellets were resuspended in lysis buffer (prepared as described in the [http://openwetware.org/wiki/Knight:Purification_of_His-tagged_proteins/Denaturing Knight protocol]) containing urea and sonicated for 15 minutes. For the western blot the samples were run on an SDSpage gel and then transferred to a nitrocellulose membrane. The detection was accomplished using the following antibodies: Anti-his biotin as primary and Streptavidin-HPR as a secondary antibody. Additional to the primary antibody we applied Anti-his HRP. |
The supernatants were run through a filter device with a 5 kDa cutoff (“centricon”). These samples were also anayzed with a western blot as described above. | The supernatants were run through a filter device with a 5 kDa cutoff (“centricon”). These samples were also anayzed with a western blot as described above. | ||
{{EPFL_2010_bottom_wrap}} | {{EPFL_2010_bottom_wrap}} | ||
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Revision as of 19:45, 26 October 2010
Materials and Methods
We grew E.Coli DH5a transformed with the plasmid C3 containing the [http://partsregistry.org/Part:BBa_K320001 immunotoxin] with the [http://partsregistry.org/Part:BBa_K320002 strong promoter]([http://partsregistry.org/Part:BBa_K320006 BBa_K320006] ). As a negative control we used cultures transformed with the C3 plasmid alone. A culture volume of 100 ml was spinned down. The pellets as well as the supernatents were used as samples for a protein analysis. The pellets were resuspended in lysis buffer (prepared as described in the [http://openwetware.org/wiki/Knight:Purification_of_His-tagged_proteins/Denaturing Knight protocol]) containing urea and sonicated for 15 minutes. For the western blot the samples were run on an SDSpage gel and then transferred to a nitrocellulose membrane. The detection was accomplished using the following antibodies: Anti-his biotin as primary and Streptavidin-HPR as a secondary antibody. Additional to the primary antibody we applied Anti-his HRP. The supernatants were run through a filter device with a 5 kDa cutoff (“centricon”). These samples were also anayzed with a western blot as described above.
