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Team:Washington/Protocols/EnzymeAssayCapD
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| - | + | ='''Enzyme Assay for CapD + CapDcp'''= | |
''Run one transpeptidase reaction and one hydrolysis reaction per enzyme (always run control for each set)'' | ''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) | + | '''Prepare 10x Master Mix with these concentrations. (10uL/rxn)''' |
*HEPES 7.4 pH (250mM) | *HEPES 7.4 pH (250mM) | ||
*1% Tween | *1% Tween | ||
*Substrate (500nM) | *Substrate (500nM) | ||
| - | Dilute Enzymes | + | '''Dilute Enzymes''' |
*Measure enzyme concentrations by protein gel band intensity or with spectrophotometer (A280) | *Measure enzyme concentrations by protein gel band intensity or with spectrophotometer (A280) | ||
*Dilute enzymes to 50nM (~0.0025mg/mL) in strip tubes | *Dilute enzymes to 50nM (~0.0025mg/mL) in strip tubes | ||
**''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 | ||
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*Pipette 10uL of 50mM L-Glu into each '''transpeptidation''' reaction 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 | + | '''Prepare Spectramax plate reader with desired settings and desired emission and excitation wavelengths''' |
*Pipette 10uL of your enzyme into each well | *Pipette 10uL of your enzyme into each well | ||
*Immediately place into Spectramax plate reader and begin reading | *Immediately place into Spectramax plate reader and begin reading | ||
Revision as of 22:34, 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
