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Caspase 8 Activity Assay Kit (by Spectrophotometry)[312]

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Price:   $68
Cat Number:  E-CK-A312
Lead Time:   7~10 days
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Introduction

Elabscience® Caspase 8 Activity Assay Kit can detect the activity of Caspase 8 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 8 (also known as FLICE, MACH or mch5) is considered to be a caspase in the upstream of apoptotic signal transduction. Caspase 8 is activated in Fas-receptor and TNFR-1-mediated apoptotic process, forming a dimer composed of P18 and p10, which further activates downstream Caspase 4, Caspase 6, Caspase 9 and Caspase 10. Caspase 8 exists in the form of prozyme in the normal state and has no activity. However, Caspase 8 is activated in the apoptotic stage and participates in the apoptotic process.

This kit is used to conjugate Caspase 8 sequence-specific peptides acetyl-Ile-Glu-Val-Asp p-nitroanilide (Ac-IETD-pNA) to yellow group p-nitroaniline (pNA). When the substrate is cut by Caspase 8, the yellow group pNA is dissociated. pNA has an absorption peak at 405 nm. Measure the OD value at 405 nm and then Caspase 8 activity can be calculated accordingly.


Components


Cat.

Products

20 Tests

50 Tests

100 Tests

Storage

   E-CK-A312A

     Lysis Buffer

3.0 mL

7.5 mL

15 mL

-20°C

   E-CK-A312B

     2 ×Reaction Buffer

1.0 mL

1.25 mLx 2

5.0 mL

-20°C

   E-CK-A312C

     Ac-IETD-pNA

100 μL

250 μL

500 μL

-20°C

   E-CK-A312D

     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-IETD-pNA[E-CK-A312C] 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 8 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 8, 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 8 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 8 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 8 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