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Calpain Activity Fluorometric Assay Kit

based on 30 citations in multiple journalsCalpain Activity Fluorometric Assay Kit305 5
Exclusively detects activated calpain
Catalog #: K240
$495.00

Product Details

Cat # +Size K240-100
Size 100 assays
Detection Method Fluorescence (Ex/Em 400/505 nm)
Species Reactivity Mammalian
Applications The kit provides a simple and convenient means for analyzing calpain activity in apoptotic and other samples. The assay is based on fluorometric detection of the cleavage of calpain substrate at 505 nm using a fluorometer or a fluorecence plate reader.
Features & Benefits • Simple one-step procedure; takes only ~2 hours
• Fast and convenient
• The kit contains both positive and negative controls. Active calpain and calpain inhibitor and antibodies are also available separately.
Kit Components • Extraction Buffer
• 10X Reaction Buffer
• Calpain Substrate Ac-LLY-AFC
• Active Calpain I (Positive Control)
• Calpain Inhibitor Z-LLY-FMK
Storage Conditions -70°C
Shipping Conditions Dry Ice
USAGE For Research Use Only! Not For Use in Humans.

Details

Activation of calpain is involved in many forms of physiological and pathological processes (e.g., apoptosis). Calpain activation requires cell membrane and Ca²ᶧ, and activated calpain is released into cytosol. The Calpain Activity Assay Kit provides optimized buffers and reagents for a convenient measurement of calpain activity. The Extraction Buffer provided with the kit specifically extracts cytosolic proteins without contaminations of cell membrane and lysosome proteases. The Extraction Buffer also prevents auto-activation of calpain during the extraction procedure. Thus, the kit detects only activated calpain in cytosol upon treatment of cells with inducers (e.g., chemicals or drugs). The fluorometric assay is based on the detection of cleavage of calpain substrate Ac-LLY-AFC. Ac-LLY-AFC emits blue light (λmax = 400 nm); upon cleavage of the substrate by calpain, free AFC emits a yellow-green fluorescence (λmax = 505 nm), which can be quantified using a fluorometer or a fluorecence plate reader. Comparison of the fluorescence intensity from a treated sample with a normal control allows determination of the changes in calpain activity.


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We have used increasing amounts of active Calpain 1 with this assay, but our readings are not linear. Why?
The most likely reason for the signal to be non-liear is the substrate saturation.
Calpains will automatically be activated by calcium as soon as the plasma membrane is broken to collect cellular proteins. Does your kit take this into consideration?
We do take this into consideration. Pro-calpain activation requires cell membrane, and after activation the active calpain is released into cytosol. 1) The Calpain kit utilizes mild detergent in the Extraction buffer to extract only activated calpain present in cytosol, but not inactivated calpain which is still associated with cell membrane. 2) To prevent the auto-activation of calpain, the Extraction buffer is supplied with optimal concentrations of EDTA and EGTA to chelate the calcium to avoid any autoactivation of calpain during the extraction procedure.
The protocol included with the kit does not describe how to perform the assay directly on cells cultured in a 96 well plate. How do I perform the assay in the plate?
It would be difficult to perform the assay directly in the same 96-well plate, as you would need to extract the activated calpain and transfer the extract into a new plate before performing the analysis.
The Calpain that we are interested in, is connected to Talin, which form along with other proteins a large focal adhesion complex that is attached to membranes (mostly plasma membrane). In the lysis procedure described in the manual that came with the assay, it says: "The extraction buffer provided with the kit specifically extracts CYTOSOLIC proteins without contaminations of cell membrane". Would you think that this lysis will be effective enough to include Calpain in complexes?
The kit is designed for detecting activated cytosolic calpain after translocate from cell membrance to cytosol. If you want to detect all calpains that associated with membrane, you can add 1% triton X-100 into the extraction buffer, which will extract the all calpains from the cells.
Since the assay comes with no "stop reaction buffer" to use before the O.D. reading - is it correct to assume that the 1h incubation @ 37C of the sample and the Calpain substrate CONSUME the enzymatic reaction? If it will take several minutes to get the color reaction to actual reading - will the color of the reaction maintain itself ?
The active enzyme is still in the reaction, and the reaction is still going on. The color will be there for quite long time. If you want to stop the reaction, you may add 1% SDS to inactivate active calpain.
If I increase number of cells, how should I adjust volume of extraction buffer?
When you are increasing the number of cells you are aiming for higher concentrations. The adjustment of the volume will defeat that purpose. Increase the amount of lysate and keep every thing else the same.
Is the Calpain inhibitor specific only calpain I?
This calpain inhibitor inhibits both calpain I and II (m calpain and mu calpain).
Can this kit be used with samples like bacteria, plants, drosophila, yeast etc?
We have optimized the kit with mammalian samples. However, theoretically these kits should work with samples from multiple species/sources. Since the optimal conditions depend on the sample type, the protocol has to be be adapted to fit the samples for efficient results. Please refer to this kits citations to see what kind of samples have been used with this kit other than mammalian samples.
Can we use a different wavelength than recommended for the final analysis?
It is always recommended to use the exact recommended wavelength for the most efficient results. However, most plate readers have flexibility in their band width of detection in increments of +/- 10 nm. Depending on this flexibility range, you can deviate from the recommended wavelengths within limits.
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 this area please contact yopur regional BioVision distributor.
Can we purchase individual components of this kit?
Yes, you can purchase any of the kits components without the whole kit. Please refer to the component Cat #s mentioned on the datasheet for ordering.
Hissa, Zhi et al. (2017) Cholesterol depletion impairs contractile machinery in neonatal rat cardiomyocytes, Sci Rep. 2017 Mar 3;7:43764.
Sun et al., GAS2–Calpain2 axis contributes to the growth of leukemic cells. Acta Biochim Biophys Sin, Oct 2015; 47: 795 - 804.
Zou et al., Phytoestrogen β-Ecdysterone Protects PC12 Cells Against MPP+-Induced Neurotoxicity In Vitro: Involvement of PI3K-Nrf2-Regulated Pathway. Toxicol. Sci., Jul 2015; 10.1093/toxsci/kfv111.
Wu et al., Calpain-Dependent Cleavage of Junctophilin-2 and T-Tubule Remodeling in a Mouse Model of Reversible Heart Failure. JAHA, Jun 2014; 3: e000527.
Chen et al., Effects of isoproterenol on aquaporin 5 levels in the parotid gland of mice in vivo. Am J Physiol Endocrinol Metab, Jan 2014; 306: E100 - E108.
For more citations of this product click here