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From 2010.igem.org

Contents 1 Andreas 1.1 IgG protease assay 2 Nina 2.1 Protein A overexpression 2.2 Protein A on Tris-gel 2.3 Glycerol Stock 3 Mimmi 3.1 SOD activity 3.2 SOD activity standard curve 3.2.1 plasmid prep 4 Johan 4.1 Protein A overexpression

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.

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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

Mimmi

SOD activity

• Start culture from ON culture
  • 40ml LB_amp
  • 400µl old culture
    • pEX.SOD
    • pEX.yCCS

• At OD=6.0, add IPTG 1mM
  • take 10ml samples at 0h, 1h and 2h
  • Spinn down and remove LB
  • Resuspend in 1ml phosphate buffer, pH=7.0
    • keep on ice!
  • Transfer to eppendorf tube

• Sonicate

SOD activity standard curve

mix	samples	blank 1	blank 3
sample solution	20
ddH2O		20	20
WST solution	200	200	200
Enzyme solution	20	20
dilution buffer			20
tot	240µl	240µl	240µl

• Incubate in 37°C for 20 min

• Measure A₄₄₀ with nanodrop

• SOD activity (inhibition %) = ((A_blank1 - A_blank3) - (A_sample - A_blank2))/(A_blank1 - A_blank3) x 100

plasmid prep

• Follow E.T.Z.N.A plasmid mini prep protocol
  • Wash two times with DNA wash buffer
  • Eluate two times in 70µl dH₂O

Johan

Protein A overexpression

The OD of two overnight cultures of protein A (different colonies) was checked, they had OD 2,8 and 2,6!

I first diluted them 4 times, but then decided that there should be too many dead cells and I diluted it further 750 µl in 50 ml LB.

I then overexpressed the cells with IPTG and then run an tris-gel, Nina has more details.