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Team:Washington/Protocols/EnzymeAssayCapD
From 2010.igem.org
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(→Enzyme Assay for CapD + CapDcp) |
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**''Make sure you have at least 30uL of your final diluted enzyme'' | **''Make sure you have at least 30uL of your final diluted enzyme'' | ||
| - | Prepare a 96-well plate | + | Prepare a 96-well plate |
*Pipette diH2O into each well (Two per enzyme) | *Pipette diH2O into each well (Two per enzyme) | ||
**Transpeptidase: 70uL | **Transpeptidase: 70uL | ||
Revision as of 21:05, 21 September 2010
Enzyme Assay for CapD + CapDcp
Run one transpeptidase reaction and one hydrolysis reaction per enzyme (always run control for each set)
Prepare 10x Master Mix with these concentrations. (10uL/rxn)
- HEPES 7.4 pH (250mM)
- 1% Tween
- Substrate (500nM)
Dilute Enzymes
- Measure enzyme concentrations by protein gel band intensity or with spectrophotometer (A280)
- Dilute enzymes to 50nM (~0.0025mg/mL) in strip tubes
- Make sure you have at least 30uL of your final diluted enzyme
Prepare a 96-well plate
- Pipette diH2O into each well (Two per enzyme)
- Transpeptidase: 70uL
- Hydrolysis: 80uL
- Pipette 10uL of 10x Master Mix into each well.
- Pipette 10uL of 50mM L-Glu into each transpeptidation reaction well.
Prepare Spectramax plate reader with desired settings and desired emission and excitation wavelengths
- Pipette 10uL of your enzyme into each well
- Immediately place into Spectramax plate reader and begin reading
Final Concentrations
- Enzyme (CapD) 5nM (0.00025mg/mL)
- Amino Acid (L-Glutamate)0mM or 5mM
- Substrate 50nM
- HEPES (7.4pH) 25mM
- 0.1% Tween
- Final Reaction Volume 100uL
