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Team:Washington/Protocols/EnzymeAssayCapD
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=='''Enzyme Assay for CapD + CapDcp'''== | =='''Enzyme Assay for CapD + CapDcp'''== | ||
| - | Run one transpeptidase reaction and one hydrolysis reaction per enzyme | + | ''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) | ||
| - | '''Prepare Hydrolysis | + | 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''' | '''Final Concentrations''' | ||
*Enzyme (CapD) 5nM (0.00025mg/mL) | *Enzyme (CapD) 5nM (0.00025mg/mL) | ||
| - | *Amino Acid (L-Glutamate) | + | *Amino Acid (L-Glutamate)0mM or 5mM |
*Substrate 50nM | *Substrate 50nM | ||
*HEPES (7.4pH) 25mM | *HEPES (7.4pH) 25mM | ||
Revision as of 21:03, 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
