Team:Stockholm/20 October 2010
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==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. | ||
+ | |||
+ | ---- | ||
+ | ==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]] | ||
+ | |||
+ | 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<sub>amp</sub> | ||
+ | **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 | ||
+ | | | ||
+ | | | ||
+ | |- | ||
+ | | ddH<sub>2</sub>O | ||
+ | | | ||
+ | | 20 | ||
+ | | 20 | ||
+ | |- | ||
+ | | WST solution | ||
+ | | 200 | ||
+ | | 200 | ||
+ | | 200 | ||
+ | |- | ||
+ | | Enzyme solution | ||
+ | | 20 | ||
+ | | 20 | ||
+ | | | ||
+ | |- | ||
+ | | dilution buffer | ||
+ | | | ||
+ | | | ||
+ | | 20 | ||
+ | |- | ||
+ | | align="right" | tot | ||
+ | | 240µl | ||
+ | | 240µl | ||
+ | | 240µl | ||
+ | |} | ||
+ | |||
+ | *Incubate in 37°C for 20 min | ||
+ | |||
+ | |||
+ | *Measure A<sub>440</sub> with nanodrop | ||
+ | |||
+ | |||
+ | *SOD activity (inhibition %) = ((A<sub>blank1</sub> - A<sub>blank3</sub>) - (A<sub>sample</sub> - A<sub>blank2</sub>))/(A<sub>blank1</sub> - A<sub>blank3</sub>) x 100 | ||
+ | |||
+ | |||
+ | [[Image:SOD_activity_standard_curve.jpg| 900px]] | ||
+ | |||
+ | |||
+ | |||
+ | ==== plasmid prep ==== | ||
+ | |||
+ | *Follow E.T.Z.N.A plasmid mini prep protocol | ||
+ | **Wash two times with DNA wash buffer | ||
+ | **Eluate two times in 70µl dH<sub>2</sub>O | ||
+ | |||
+ | =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. | ||
+ | |||
+ | {{Stockholm/Footer}} |
Latest revision as of 18:51, 27 October 2010
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:
- 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
Mimmi
SOD activity
- Start culture from ON culture
- 40ml LBamp
- 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 %) = ((Ablank1 - Ablank3) - (Asample - Ablank2))/(Ablank1 - Ablank3) 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.
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