Team:Washington/Protocols/MMAssay
From 2010.igem.org
(Difference between revisions)
(→Michaelis-Menten Assay) |
|||
(6 intermediate revisions not shown) | |||
Line 27: | Line 27: | ||
'''Prepare a 10x Master Mix with these concentrations (10uL/rxn)''' | '''Prepare a 10x Master Mix with these concentrations (10uL/rxn)''' | ||
+ | |||
Make one for each enzyme | Make one for each enzyme | ||
*HEPES 7.4 pH (250mM) | *HEPES 7.4 pH (250mM) | ||
*1% Tween | *1% Tween | ||
*Purified Enzyme (50nM) | *Purified Enzyme (50nM) | ||
+ | **''Dilute enzyme in 1x HEPES/Tween buffer'' | ||
''Note: Make one without enzyme for blank'' | ''Note: Make one without enzyme for blank'' | ||
- | + | '''Prepare Substrate''' | |
- | + | ||
- | + | ||
*Prepare 10x substrate (10uM) in one well of a 12-well strip tube | *Prepare 10x substrate (10uM) in one well of a 12-well strip tube | ||
- | *Do half concentration serial dilutions until 11th well. | + | *Do half concentration serial dilutions (equal parts diH2O and substrate) until 11th well. |
*Leave the 12th well with no substrate | *Leave the 12th well with no substrate | ||
+ | ''Final Concentrations of Substrate (Left to Right in uM): '' | ||
+ | |||
+ | ''10, 5, 2.5, 1.25, 0.625, 0.3125, 0.15625, 0.078125, 0.0390625, 0.01953125, 0.009765625'' | ||
'''1.1x Master Mix''' | '''1.1x Master Mix''' | ||
*Put equal parts 10x Master Mix and 10x L-Glu eppendorf tube (Referred to as L-Glu Master Mix) | *Put equal parts 10x Master Mix and 10x L-Glu eppendorf tube (Referred to as L-Glu Master Mix) | ||
*Put equal parts 10x Master Mix and diH2O in an eppendorf tube (Referred to as diH2O Master Mix) | *Put equal parts 10x Master Mix and diH2O in an eppendorf tube (Referred to as diH2O Master Mix) | ||
- | *Fill each tube with diH2O | + | *Fill each tube with diH2O (70uL/rxn) |
'''Prepare a 96-well plate for transpeptidation''' | '''Prepare a 96-well plate for transpeptidation''' | ||
Line 55: | Line 58: | ||
*Make one row of blank (1.1x diH2O Master Mix without enzyme) | *Make one row of blank (1.1x diH2O Master Mix without enzyme) | ||
- | Final Concentrations | + | '''Prepare Spectramax plate reader with desired settings''' |
+ | *Pipette 10uL of Substrate into each well. (Column 1 from well 1 of strip tube, Column 2 from well 2 of strip tube, etc.) | ||
+ | *Immediately place into Spectramax plate reader and begin reading | ||
+ | |||
+ | '''Final Concentrations''' | ||
*Enzyme (CapD) - 5nM (0.00025mg/mL) | *Enzyme (CapD) - 5nM (0.00025mg/mL) | ||
*Amino Acid (L-Glutamate) - 0mM or 5mM | *Amino Acid (L-Glutamate) - 0mM or 5mM | ||
Line 64: | Line 71: | ||
Final Reaction Volume 100uL | Final Reaction Volume 100uL | ||
+ | ''Substrate is 5-FAM-(D-gamma-Glu)5-K(QXL520-NH2 from AnaSpec'' | ||
+ | ''Product produced from reaction is 5-FAM-(D-gamma-Glu)5-NH2 from AnaSpec'' | ||
<!---------------------------------------PAGE CONTENT GOES ABOVE THIS----------------------------------------> | <!---------------------------------------PAGE CONTENT GOES ABOVE THIS----------------------------------------> | ||
<div style="text-align:center"> | <div style="text-align:center"> | ||
'''← [[Team:Washington/Protocols|Back to Lab Protocols]]''' | '''← [[Team:Washington/Protocols|Back to Lab Protocols]]''' | ||
| | ||
- | |||
{{Template:Team:Washington/Templates/Footer}} | {{Template:Team:Washington/Templates/Footer}} |
Latest revision as of 20:31, 27 October 2010
Michaelis-Menten Assay
Prepare a 10x Master Mix with these concentrations (10uL/rxn)
Make one for each enzyme
- HEPES 7.4 pH (250mM)
- 1% Tween
- Purified Enzyme (50nM)
- Dilute enzyme in 1x HEPES/Tween buffer
Note: Make one without enzyme for blank
Prepare Substrate
- Prepare 10x substrate (10uM) in one well of a 12-well strip tube
- Do half concentration serial dilutions (equal parts diH2O and substrate) until 11th well.
- Leave the 12th well with no substrate
Final Concentrations of Substrate (Left to Right in uM):
10, 5, 2.5, 1.25, 0.625, 0.3125, 0.15625, 0.078125, 0.0390625, 0.01953125, 0.009765625
1.1x Master Mix
- Put equal parts 10x Master Mix and 10x L-Glu eppendorf tube (Referred to as L-Glu Master Mix)
- Put equal parts 10x Master Mix and diH2O in an eppendorf tube (Referred to as diH2O Master Mix)
- Fill each tube with diH2O (70uL/rxn)
Prepare a 96-well plate for transpeptidation
- Pipette 90uL of 1.1x L-Glu Master Mix for one each enzyme across a row for the transpeptidation reaction
- Repeat for each enzyme
- Make one row of blank (1.1x L-Glu Master Mix without enzyme)
Prepare a 96-well plate for hydrolysis
- Pipette 90uL of 1.1 diH2O Master Mix for each different enzyme in each new row
- Repeat for each enzyme
- Make one row of blank (1.1x diH2O Master Mix without enzyme)
Prepare Spectramax plate reader with desired settings
- Pipette 10uL of Substrate into each well. (Column 1 from well 1 of strip tube, Column 2 from well 2 of strip tube, etc.)
- 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 - Variable Concentration
- HEPES (7.4pH) - 25mM
- 0.1% Tween
Final Reaction Volume 100uL
Substrate is 5-FAM-(D-gamma-Glu)5-K(QXL520-NH2 from AnaSpec
Product produced from reaction is 5-FAM-(D-gamma-Glu)5-NH2 from AnaSpec