Team:Stockholm/20 October 2010

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(Difference between revisions)
(→Andreas)
(→IgG protease assay)
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'''Procedures'''<br />
'''Procedures'''<br />
See protocols page.
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:
 +
 +
[[Image:Ls.jpg]]
 +
 +
Ladder: PageRuler Unst. Protein Ladder Fermentas
 +
 +
[[Image:Mlös1.jpg|200px]]
 +
 +
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

Revision as of 14:50, 24 October 2010

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Contents

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:

  1. Protein extract is prepared from IPTG-induced cells overexpressing IdeS (IgG protease).
  2. α-mouse IgG-peroxidase goat IgG secondary antibodies (Sigma-Aldrich) are bound to mouse IgG-Agarose (Sigma-Aldrich) beads.
  3. Excess/unbound secondary antibody are removed by washing with PBS.
  4. 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.
  5. After spinning down agarose beads, supernatant is collected.
  6. 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:

Ls.jpg

Ladder: PageRuler Unst. Protein Ladder Fermentas

Mlös1.jpg

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
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