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Cholesterol Esterase - Assay

Source:
Porcine Pancreas
CAS:
9026-00-0
EC:
3.1.1.13
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Cholesterol Esterase Assay

Method

The initial rate of hydrolysis of cholesterol ester is determined by following the increase in absorbance at 505 nm. One unit hydrolyzes 1 μmole of cholesterol ester per minute at 37°C and pH 7.0 under the specified conditions.

Reagents
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  • 0.1 M Phosphate buffer, pH 7.0
  • 0.7 mM 4-aminoantipyrine in 0.1 M Phosphate buffer, pH 7.0. Discard after 8 hours.
  • 5.0 mM Phenol in 0.1 M Phosphate buffer, pH 7.0. Discard after 8 hours. Use only fresh, white phenol crystals.
  • 5% Triton X-100. Store at 4°C.
  • Sodium Cholate: Dissolve 3.51 g Sodium cholate in 10.0 ml reagent grade water. Discard after 8 hours.
  • Cholesterol Oxidase: Dissolve 28.5 units cholesterol oxidase/ml in reagent grade water. Discard after 8 hours.
  • Peroxidase: 8200 u/ml in reagent grade water. Discard after 8 hours.
  • Cholesteryl Acetate. Dissolve 220 mg Cholesteryl Acetate (Eastman) in 50.0 ml Isopropanol (ACS Grade). Stir vigorously to dissolve. Store at 4° C.
Enzyme

Dissolve one mg/ml in 0.1 M Phosphate buffer and then prepare further dilutions (usually 2-3X) to achieve a rate of approximately 0.045/minute.

Procedure

Adjust spectrophotometer to 505 nm and 37°C. Prepare master batch, sufficient for 5 tests, by pipetting into a beaker:

0.7 mM 4-aminoantipyrine 7.0 ml
5.0 mM Phenol 7.0 ml
Sodium Cholate 0.5 ml
5% Triton X-100 0.5 ml
Cholesterol Oxidase 0.25 ml
Peroxidase 0.05 ml

Mix gently. Stored at 4°C, master batch is stable for 8 hours.

Pipette into test and blank cuvettes 3.0 ml master batch. Add 0.10 ml cholesterol acetate. Mixture should be used within 1 hour. Equilibrate temperature to 37°C. Add to test cuvettes, 0.1 ml enzyme dilution and 0.1 ml reagent grade water to the blank. If the blank rate exceeds 0.005 ΔA per minute, reincubate for 5 more minutes. Determine ΔA505 of test and blank cuvettes using the linear portion of the first 5 minutes of the reaction. Subtract blank rates, if any.

Calculation

Cholesterol Esterase Products

Description
Activity
Code
Cat. #
Size
Price
Description
Cholesterol Esterase
Source:
Porcine Pancreas
A lyophilized powder chromatographically purified to minimize protease contamination.
Store at -20°C.
…
Ice Pack required
Activity
≥300 units per gram dry weight
Code
CEPM
Minimum order quantity
10000
LS004105
Bulk
---
The minimum amount for bulk packaging/pricing for this product is 10000 x 1 un. Please contact custservice@worthington-biochem.com to request a quote for smaller amounts.
LS004104
500 un
$161.00
LS004103
100 un
$45.00