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Caspase 3 Activity Assay Kit (by Spectrophotometry)[311]

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Price:   $68
Cat Number:  E-CK-A311
Lead Time:   7~10 days
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Introduction

Elabscience® Caspase 3 Activity Assay Kit can detect the activity of Caspase 3 in cells or tissue lysis.


Detection principle

Caspase (Cysteine-requiring Aspartate Protease) is a protease family that plays an important role in the process of apoptosis. Caspase 3 (also known as CPP32, Yama, or apopain) is a member of the CED-3 subfamily of Caspases and is one of the critical enzymes of apoptosis. Caspase 3 is the most studied Caspase in mammalian cells. Caspase 3 can be used to cut proCaspase2, 6, 7, 9, and cut specifically many substrates of Caspase directly, including PPAR (poly ADP-ribose), the inhibitor of Caspase-activated deoxyribonuclease (ICAD), gelsolin and fodrin. Protein shearing mediated by Caspase 3 are important parts of the molecular mechanism of apoptosis. In addition, Caspase 3 also plays a key role in the process of nuclear apoptosis including chromatin condensation and DNA fragmentation as well as cell blebbing. Caspase 3 exists in the form of prozyme in the normal state and has no activity. However, during the apoptosis stage, activated Caspase 3 consists by two large subunits and two small subunits, which cleavages the corresponding substrate of endochylema or cytoplasmic nuclear and eventually leads to apoptosis.

This kit is used to conjugate Caspase 3 sequence-specific peptides acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA) to yellow group p-nitroaniline (pNA). When the substrate is cut by Caspase 3, the yellow group pNA is dissociated. pNA has an absorption peak at 405 nm. Measure the OD value at 405 nm and then

Caspase 3 activity can be calculated accordingly.


Components


Cat.

Products

20 Tests

50 Tests

100 Tests

Storage

E-CK-A311A

Lysis Buffer

3.0 mL

7.5 mL

15mL

-20°C

E-CK-A311B

2 ×Reaction Buffer

1.0 mL

1.25 mLx2

5.0 mL

-20°C

E-CK-A311C

Ac-DEVD-pNA

100 μL

250 μL

500 μL

-20°C

E-CK-A311D

DTT

50 μL

100 μL

150 μL

-20°C

Manual

 One Copy



Reagents not included

PBS, Protein Quantitative Kit (Bradford, optional).


Storage

Store at -20°C. Ac-DEVD-pNA[E-CK-A311C] should be stored in dark. Avoid freeze / thaw cycles.


Note

1.  For maximal assay performance, this reagent should be used within 12 months. Avoid freeze / thaw cycles.

2.  There is reducing agent (DTT) in the Lysis Buffer,It is recommended to Elabscience® Total Protein (TP) Colorimetric Assay Kit (Coomassie Brilliant Blue Method)(It is recommended to use Elabscience® E-BC-K168-S) to measure the protein concentration instead of BCA method.

3.  It has been reported that the activity of Caspase 3 can’t be detected in few types of apoptosis, which may be due to the existence of a mechanism independent of the activation of Caspase 3, and other signaling pathways in the mechanism of apoptosis need to be considered. In this case, there is no significant change in the activity of Caspase 3 by using this kit.

4.  Lysis Buffer in this kit can be used in other Caspase activity assay kits by Elabscience®. And the protein sample can test other Caspase activity.

5.  pNA (4-nitroaniline) is toxic. Please be careful when operating, and pay attention to effective protection to avoid direct contact with human body or inhalation. pNA solidifies at lower temperatures and sticks to the bottom, wall or cap of centrifugal tube. It can be incubated in water bath at 20~25°C for a short time until it is completely melted.

6.  When the activity of Caspase 3 in the sample is very low. Confirm whether the phenomenon of apoptosis is obvious or not firstly. If the apoptosis is obvious and we confirm that Caspase can be activated, please adjust the time of apoptosis and find a time point which Caspase 3 activation is stronger. And then repeat the test.

7.  This kit is for research use only. For your safety and health, please wear lab clothes and gloves. Instructions should be followed strictly, changes of operation may result in unreliable results.

If you have any question about the product, please feel free to contact us or leave a message