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Team:Stockholm/20 October 2010
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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.
Nina
Protein A overexpression
I induced with IPTG an overnight cuture of 12 ml Protein A.His (N terminal) inserted in the overexpression vector (pEX).
- Samples were taken at 0, 1, 2 & 3 h.
- Pelleted by 1 min of centrifugation at 13 000 rpm.
- Resuspened with 50 ul water and added additional 50 ul of RDSB (Sample buffer with DTT). All samples were stored in the freezer until the induction of 3 hours was done.
- Sonicated for 40 seconds.
- Heated at 95 °C for 5 min.
- Centrifuged 30 seconds at 13 000 rpm.
- Supernatants were added in a gel.
Protein A on Tris-gel
I found out that protein A Z domain that I am working with is not possible to observe on an usual polyacrylamide gel since it is very small (7kDa), I would need to run the overexpression samples on a Tris-gel.
The gel I used was a Tris-gel 10-20% from invitrogen.
Arragement on gel:
After the gel was done I left in a box on shake in coomassie blue staining overnight.
Glycerol Stock
I made a glycerol stock of Protein A.His (N terminal) in the pEX vector.
- 400 ul gycerol
- 800 ul overnight sample
