Description :
The Calcineurin Cellular Activity Assay Kit is a complete colorimetric assay kit for measuring cellular calcineurin (PP2B) phosphatase activity. It employs a convenient 96-well microtiter-plate format with all reagents necessary for measuring calcineurin (PP2B) phosphatase activity in tissue/cellular extracts PLUS, human recombinant calcineurin5 is included as a positive control! The RII phosphopeptide substrate, supplied with this kit, is the most efficient and outstanding peptide substrate known for calcineurin. The detection of free-phosphate released is based on the classic Malachite green assay and offers the following advantages: Nonradioactive; convenient 1-step detection; excellent sensitivity.
Stability :
Store all components except the microtiter plate at -70°C for the highest stability. The calcineurin enzyme component must be handled particularly carefully in order to retain maximal enzymatic activity. Thaw it quickly in a RT water bath or by rubbing between fingers, then immediately store on an ice bath. The remaining unused enzyme should be quickly refrozen by placing at -70°C. To minimize the number of freeze/thaw cycles, aliquot the calcineurin into separate tubes and store at -70°C.
Kit Components :
1. Calcineurin enzyme (human, recombinant)
FORM: 50 mM Tris, pH 7.5, 100 mM NaCl, 6 mM MgCl2, 5 mM DTT, 0.025% NP-40, 0.5 mM CaCl2.
1 U=1 pmol/min at 30°C.
STORAGE: -70°C; AVOID FREEZE/THAW CYCLES!
QUANTITY: 500 U
2. Calmodulin (human, recombinant)
FORM: 25 µM in dH2O
STORAGE: -70°C
QUANTITY: 100 µL
3. Substrate (RII phosphopeptidee, sequence Asp-LeuAsp-Val-Pro-Ile-Pro-Gly-Arg-Phe-Asp-Arg-Arg-Val-pSer-Val-Ala-Ala-Glu; MW=2192.0)
FORM: 1.5 mg net peptide/vial
STORAGE: -20°C
QUANTITY: 1 x 1.5 mg
4. 2x assay buffer
FORM: 100 mM Tris, pH 7.5, 200 mM NaCl, 12 mM MgCl2, 1mM DTT, 0.05% NP-40, 1 mM CaCl2
STORAGE: -70°C
QUANTITY: 20 mL
5. 2x EGTA Buffer
FORM: 100 mM Tris, pH 7.5, 20 mM EGTA, 200 mM NaCl, 12 mM MgCl2, 1 mM DTT, 0.05% NP-40
STORAGE: -70°C
QUANTITY: 1 mL
6. Lysis Buffer
FORM: 50 mM Tris, pH 7.5, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.2% NP-40
STORAGE: -70°C
QUANTITY: 1 X 40 mL
7. Protease inhibitor cocktail
FORM: Solid tablet
STORAGE: 4°C
QUANTITY: 2 tablets
8. Biomol Green Reagent
FORM: Liquid in screw-cap plastic bottle.
STORAGE: 4°C
QUANTITY: 20 mL
9. Phosphate standard
FORM: 80 µM in dH2O
STORAGE: -70°C
QUANTITY: 0.5 mL
10. Okadaic acid
HARMFUL! AVOID ALL FORMS OF CONTACT.
FORM: 5 µM in 2X Assay Buffer
STORAGE: -70°C.
QUANTITY: 325 µL
11. Desalting column and resin
FORM: 5 mL polypropylene disposable column and P6 DG desalting resin
STORAGE: RT. After rehydration store at 4°C.
QUANTITY: 1 column and 1 g of resin
12. ½-Volume microplate
2 clear, 96-well
STORAGE: Room temperature
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