Elastase Assay

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Elastase Assay

Method

Bieth et al. (1974) have described an excellent assay using succinyl-(L-alanine)₃-p-nitroanilide. Other suggested assays have been described: Keller and Mandl (1971), Feinstein et al. (1973), and Bieth and Meyer (1973). Schumacher and Schill (1972) describe a radial diffusion assay. The assay used in this laboratory is as follows:

Method: Derived from that of Feinstein et al. (1973) and using the more soluble substrate of Bieth et al. (1974). An increase in absorbance at 410 nm results from the hydrolysis of N-succinyl-L-Ala-L-Ala-L-Ala-p-nitroanilide (Suc Ala₃NA). One unit hydrolyzes one micromole of Suc Ala₃NA per minute at 25°C and pH 8.0 under the specified conditions.

Reagents

0.1 M Tris buffer, pH 8.0
0.0044 M Suc Ala₃NA substrate dissolved in Tris buffer. (2 mg/ml)

Enzyme

Prepare a one mg/ml solution in Tris buffer. Immediately before use dilute further to obtain a rate of 0.02-0.04 ΔA/minute.

Procedure

Adjust the spectrophotometer to 410 nm and 25°C.

Pipette into each cuvette as follows:

Tris buffer 2.7 ml

Enzyme 0.1 ml

Mix and incubate in the spectrophotometer 4-5 minutes to achieve temperature equilibration. To test cuvette add 0.2 ml of substrate, mix, and record increase A₄₁₀ for 3-5 minutes. Calculate ΔA/minute from the linear portion of the curve.

Calculation

\frac{\text{Units}}{\text{mg}} = \frac{\frac{\Delta \text{A}_{410}}{\text{min}}}{\frac{\text{8.8 x mg enzyme}}{\text{ml reaction mixture}}}

Elastase Products

Description

Elastase, Purified

Source:

Porcine Pancreas

Chromatographically purified. A lyophilized powder.

Store at 2-8°C.

…

Ice Pack required

Activity

≥8 units per mg protein

Certificate of Analysis

Code

ESFF

Product details

LS006363

5 mg

$82.00

LS006365

20 mg

$240.00

LS006367

Bulk

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Description

Elastase, Lyophilized

Source:

Porcine Pancreas

Two times crystallized, (Code: ESL), supplied as a dialyzed, lyophilized powder. The enzyme should be 0.22 micron filtered after reconstitution and prior to use. Suitable for the isolation of Type II lung cells.

Store at 2-8°C.

…

Activity

≥3 units per mg protein

Certificate of Analysis

Code

ESL

Product details

LS002290

25 mg

$55.00

LS002292

100 mg

$156.00

LS002294

1 gm

$1,125.00

LS002298

Bulk

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Description

Elastase, Suspension

Source:

Porcine Pancreas

Two times crystallized. Supplied as an aqueous suspension. This preparation must be diluted to dissolve the enzyme. The diluted enzyme should be 0.22 micron filtered before use. Suitable for the isolation of Type II lung cells.

Store at 2-8°C. DO NOT FREEZE.

…

Ice Pack required

Activity

≥3 units per mg protein

Certificate of Analysis

Code

Product details

LS002274

25 mg

$52.00

LS002279

100 mg

$141.00

LS002280

1 gm

$1,065.00

LS002276

Bulk

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Elastase - Assay

Elastase Assay

Elastase Products