"
Team:Stockholm/20 October 2010
From 2010.igem.org
(Difference between revisions)
(New page: {{Stockholm/Top2}} ==Andreas==) |
(→Andreas) |
||
| Line 1: | Line 1: | ||
{{Stockholm/Top2}} | {{Stockholm/Top2}} | ||
==Andreas== | ==Andreas== | ||
| + | ===IgG protease assay=== | ||
| + | Me and Elisabeth designed an assay for investigating the IgG protase activity of our BioBrick. In short, the idea behind the assay is as follows: | ||
| + | |||
| + | # Protein extract is prepared from IPTG-induced cells overexpressing IdeS (IgG protease). | ||
| + | # α-mouse IgG-peroxidase goat IgG secondary antibodies (Sigma-Aldrich) are bound to mouse IgG-Agarose (Sigma-Aldrich) beads. | ||
| + | # Excess/unbound secondary antibody are removed by washing with PBS. | ||
| + | # Protein extract is added and left to incubate ON. | ||
| + | #* This step allows for digestion of IgG-peroxidase, thereby releasing the peroxidase from the agarose beads. | ||
| + | # After spinning down agarose beads, supernatant is collected. | ||
| + | # Peroxidase substrate is added to identify presence of released peroxidase in the supernatant. | ||
| + | |||
| + | '''Procedures'''<br /> | ||
| + | See protocols page. | ||
Revision as of 17:30, 21 October 2010
| Home | Project Idea | Lab Work | Results | Modelling | Team | Follow us on  and 
| 2010.igem.org 
|
Andreas
IgG protease assay
Me and Elisabeth designed an assay for investigating the IgG protase activity of our BioBrick. In short, the idea behind the assay is as follows:
- Protein extract is prepared from IPTG-induced cells overexpressing IdeS (IgG protease).
- α-mouse IgG-peroxidase goat IgG secondary antibodies (Sigma-Aldrich) are bound to mouse IgG-Agarose (Sigma-Aldrich) beads.
- Excess/unbound secondary antibody are removed by washing with PBS.
- Protein extract is added and left to incubate ON.
- This step allows for digestion of IgG-peroxidase, thereby releasing the peroxidase from the agarose beads.
- After spinning down agarose beads, supernatant is collected.
- Peroxidase substrate is added to identify presence of released peroxidase in the supernatant.
Procedures
See protocols page.
