Recombinant B. pertussis Adenylate Cyclase Protein, CF
Recombinant B. pertussis Adenylate Cyclase Protein, CF Summary
Product Specifications
Gln2-Arg399, with an N-terminal Met and 6-His tag
Analysis
Product Datasheets
Carrier Free
CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.
8270-AC
| Formulation | Supplied as a 0.2 μm filtered solution in Tris and NaCl. |
| Shipping | The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below. |
| Stability & Storage: | Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
|
Assay Procedure
- Assay Buffer: 25 mM Tris, 10 mM MgCl2, 50 µM CaCl2, pH 7.0
- Recombinant Bacterial Adenylate Cyclase (rB.Adenylate Cyclase) (Catalog # 8270-AC)
- Recombinant Yeast Inorganic Pyrophosphatase/PPA1 (ryPPA1) (Catalog # 8088-PP)
- Calmodulin (Sigma, Catalog # P1431), 0.168 mg/mL stock in 20 mM Tris, pH 7.5, 10 mM MgCl2, 1 mg/mL BSA, and 0.1 mM CaCl2
- ATP (Sigma, Catalog # A7699), 10 mM stock in deionized water
- Malachite Green Phosphate Detection Kit (Catalog # DY996)
- 96-well Clear Plate (Catalog # DY990)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Dilute 1 M Phosphate Standard by adding 10 µL of the 1 M Phosphate Standard to 990 µL of deionized water for a 10 mM stock. Continue by adding 10 µL of the 10 mM Phosphate stock to 990 µL of Assay Buffer for a 100 µM stock. This is the first point of the standard curve.
- Continue standard curve by performing six one-half serial dilutions of the 100 µM Phosphate stock in Assay Buffer. The standard curve has a range of 0.078 to 5.0 nmol per well.
- Prepare Reaction Mixture containing 1 mM ATP and 10 µg/mL ryPPA1 in Assay Buffer.
- Dilute Calmodulin to 0.0667 µg/mL in Assay Buffer.
- Dilute rB.Adenylate Cyclase to 0.02 µg/mL in Assay Buffer.
- Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
- Load 15 µL of the 0.02 µg/mL rB.Adenylate Cyclase into empty wells of the plate.
- Add 15 µL of the 0.0667 µg/mL Calmodulin to wells containing enzyme, excluding the standard curve and curve blank. Create a Control by replacing Calmodulin with Assay Buffer.
- Start the reactions by adding 20 µL of Reaction Mixture all the wells, excluding the standard curve and curve blank.
- Seal the plate and incubate at room temperature for 10 minutes.
- Add 30 µL of the Malachite Green Reagent A to all wells.
- Add 100 µL of deionized water to all wells. Mix briefly.
- Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
- Read plate at 620 nm (absorbance) in endpoint mode.
- Calculate specific activity:
|
Specific Activity (nmol/min/µg) = |
Phosphate released* (nmol) |
| Incubation time (min) x amount of enzyme (µg) x 2 |
*Derived from the phosphate standard curve using linear or 4-parameter fitting and adjusted for Control.
Per Reaction:- rB. Adenylate Cyclase: 0.0003 µg
- ryPPA1: 0.2 µg
- Calmodulin: 0.001 µg
- ATP: 0.4 mM
Reconstitution Calculator
Background: Adenylate Cyclase
B. pertussis Adenylate Cyclase is an enzymatic toxin that converts ATP to 3’-5’cyclic AMP and plays a role in the pathogenesis of whooping cough (1). The Km for ATP of the enzyme is 0.6 mM (2). The enzyme activity is calmodulin (CaM) dependent with a Kd for CaM of 0.2 nM. The protein is synthesized as a large bifunctional precursor of 1706 amino acid residues, endowed with Adenylate Cyclase and haemolytic activity (3). The enzyme corresponds to amino acid 1 to 399 and is released from the precursor as a 43 kD fragment. The N-terminal portion (residues 1-235/237) harbors the catalytic site, whereas the C-terminal portion (residues 235/237-399) corresponds to the main CaM-binding domain of the enzyme (4).
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