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Product Details
| Cat # +Size | K360-100 |
|---|---|
| Size | 100 assays |
| Detection Method | Fluorescence (Ex/Em 335-355/ 495-510 nm) |
| Species Reactivity | Mammalian |
| Applications | Convenient fluorescence method for detecting β-secretase activity in biological and purified samples. |
| Features & Benefits | • Simple procedure; takes ~ 1 hour • Fast and convenient • The assay is sensitive, stable and high-throughput adaptable. |
| Kit Components | • β-Secretase Extraction Buffer • β-Secretase Reaction Buffer (2X) • β-Secretase Substrate (in DMSO) • Active β-Secretase (Lyophilized) • β-Secretase Inhibitor (in DMSO) |
| Storage Conditions | -80°C |
| Shipping Conditions | Dry Ice |
| USAGE | For Research Use Only! Not For Use in Humans. |
Details
β-Secretase has been implicated to be an excellent target for anti-amyloid therapy for the treatment of Alzheimer’s disease. The β-Secretase activity Assay Kit provides a convenient fluorescence method for detecting β-secretase activity in biological and purified samples. The assay utilizes a secretase-specific peptide conjugated to two reporter molecules EDANS and DABCYL. In the uncleaved form, the fluorescent emissions from EDANS are quenched by the physical proximity of the DABCYL moiety. Cleavage of the peptide by secretase physically separates EDANS and DABCYL allowing for the release of a fluorescent signal. The level of secretase enzymatic activity in samples is proportional to the level of fluorescence intensity.
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Our customer purchased the kit. She collected about 5-6 million cells, add 100 ul extraction buffer, but after incubate cell lysate on ice for 10 minutes and centrifuge at 10,000x g for 5 minutes. there are too much proteins to collect the supernatant. So I wonder whether the cell was too much? And if she add more extraction buffer ,the result was getting better? And would you give me some advise about this issue?
Thank you for your inquiry regarding BioVision's products. Two suggestions:
1. When collecting cells, make sure remove all the medium as much as possible. Spin second time if necessary. Then, the extraction buffer can lyse the cells more efficiently.
2. Since cell size are quite different, the customer may increase the volume of extraction buffer to 200ul, so the extraction will be more complete.
1. When collecting cells, make sure remove all the medium as much as possible. Spin second time if necessary. Then, the extraction buffer can lyse the cells more efficiently.
2. Since cell size are quite different, the customer may increase the volume of extraction buffer to 200ul, so the extraction will be more complete.
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 kit's citations to see what kind of samples have been used with this kit other than mammalian samples.
Can we use frozen samples with this assay?
Fresh samples are always preferred over frozen samples. However, frozen samples can also be used, provided, they were frozen right after isolation, were not freeze thawed multiple time (for which we recommend aliquoting the samples before freezing) and have been frozen for relatively short periods.
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.
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 this area please contact your regional BioVision distributor.
What is the shelf life of this kit?
This kit is good for 12 months from the date of shipment in the unopened form when stored at the appropriate temperature and appropriate conditions. After opening and reconstitution, some of the components in this kit are good for 2 months at -20°C. Please refer to the datasheet for storage information and shelf life of each of the components.
Why are my standard curve values lower than those shown on the datasheet?
There are multiple factors which influence the signals like the incubation times, room temperature, handling etc. In general, to increase the value of the standards, you can increase the incubation time. As long as the standard curve is linear, it should be fine to use, since all of your samples will also be measured under the same conditions on this curve.
How do I normalize my samples against protein concentration
You can use a protein quantitation assay on the supernatants you get from cell/tissue lysates or with any other liquid sample in the assay buffer.
Can we purchase individual components of this kit?
Yes, you can purchase any of the kit's components without the whole kit. Please refer to the component Cat #s mentioned on the datasheet for ordering.
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.
Should I make a standard curve for every expt I do, or is one curve/kit enough?
Yes, I would strongly recommend you to do the standards every time you do the expt. There is always a chance that something was done differently that day and we do not want any conditions to differ between standards and samples.
May I check with you whether the extraction buffer of K360-100 is compatible with the BCA protein assay? Does the extraction buffer have DTT?
The extraction buffer does not have DTT. But it has EDTA and detergent, which is at a minimal level and will not interfere with the assay. Therefore, you can do BCA assay with the K360 extraction buffer.
Xing, Hongxia et al. (2016) Upregulation of microRNA-206 enhances lipopolysaccharide-induced inflammation and release of amyloid-β by targeting insulin-like growth factor 1 in microglia, Mol Med Rep. 2016 Aug;14(2):1357-64.
Shimizu H et al (2008) Mol. Cell Biol. 28: 3663 - 3671.
Gamerdinger M et al (2007) Mol. Pharmacol. 72: 141-151.
Kern, A., et al. (2006) J. Biol. Chem. 281: 2405-2413.
Feyt, C., et al. (2005) J. Biol. Chem. 280: 33220-33227.
For more citations of this product click here
