Product Details
| abID | ab39707 |
|---|---|
| Detection Method | Fluorecence (400 nm excitation filter and 505 nm emission filter) |
| Species Reactivity | Mammalian |
| Applications | Detect early/middle stages of apoptosis; differentiate apoptosis from necrosis. |
| Features & Benefits | • Simple one-step procedure; takes 1-2 hours • Fast and convenient • Comparison of the fluorescence of AFC from an apoptotic sample with an uninduced control allows determination of the fold increase in caspase-6 activity. |
| Kit Components | • Cell Lysis Buffer • 2X Reaction Buffer • VEID-AFC (1 mM) • DTT (1M) |
| Storage Conditions | -20°C |
| Shipping Conditions | Gel Pack |
| USAGE | For Research Use Only! Not For Use in Humans. |
Details
Activation of ICE-family proteases/caspases initiates apoptosis in mammalian cells. The Caspase-6 Fluorometric Assay Kit provides a simple and convenient means for assaying the activity of caspases that recognize the sequence VEID. The assay is based on detection of cleavage of substrate VEID-AFC (AFC: 7-amino-4-trifluoromethyl coumarin). VEID-AFC emits blue light (λmax = 400 nm); upon cleavage of the substrate by Mch2 or related caspases, free AFC emits a yellow-green fluorescence (λmax = 505 nm), which can be quantified using a fluorometer or a fluorecence microtiter plate reader.
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Do the protease inhibitor that I added will interfere with the assay?
Protease inhibitors will interefere with the assay. Caspase 6 is a cysteine protease and can be inhibited by most protease inhibitor cocktails. The assay is based on the catalytic clevage of the substrate VEID-AFC by Caspase-6. Inhibition of caspase 6 would thus interfere with the measurement.
Can you test plasma samples?
The kit is good for cell and tissue lysates only and not for plasma samples.
What is DTT for?
A reducing environment is essential for caspases to stay in solution and to be active. DTT helps in that process.
How should tissue lysates be prepared?
For tissues, homogenize 10 mg of tissue in 200 ul of lysis buffer. Spin out debris and add 50 ul of supernatant into a well of the 96-well plate then proceed with the protocol.
What is the exact volume of sample required for this assay?
There is no specific volume we can recommend for the amount any sample to be used since it is completely sample concentration and quality based. You have to do a pilot expt with multiple sample volumes to determine the optimal volume which gives a reading within the linear range of the standard curve. Please refer to the citations for this product to see what other clients have used with similar sample types.
Do you have trial sizes of this kit?
Unfortunately, we do not have trial sizes of this kit available. However, if you are based in the US or Canada, we will give you a 10% off list price introductory discount on its purchase price. If you are based out of the United States, please contact your regional BioVision distributor.
Can we purchase individual components of kits?
You can purchase any of the kit’s components without the whole kit. Please call us or email us and refer to the component Cat #s mentioned on the datasheet for ordering.
What is the shelf life of your kits?
The expiry date is typically 1 year from the date of shipment. Please refer to the product datasheet for more details.
Can we use an alternate buffer for sample preparation (cell lysis, sample dilutions etc)?
Our assay buffers are optimized for the reactions they are designed for. They not only contain some detergents for efficient lysis of your cells/tissue, but also contain some proprietary components required for the further reactions. Therefore, we highly recommend using the buffers provided in the kit for the best results.
Chen T-H et al (2008) Clin. Cancer Res. 14: 4250- 4258.
Deane, A, Jennifer., et al. (2004) J. Immunol. 172:6615-6625.
Coletti, D., et al. (2002) EMBO J. 21:631-642.
For more citations of this product click here
