Overview

  • Product name
    Beta Secretase Activity Assay Kit (Fluorometric)
    See all Beta secretase kits
  • Detection method
    Fluorescent
  • Sample type
    Urine, Serum, Plasma, Other biological fluids, Cell Lysate, Cell culture media, Tissue Lysate
  • Assay type
    Enzyme activity
  • Assay time
    1h 00m
  • Species reactivity
    Reacts with: Other species, Mammals
  • Product overview

    Beta Secretase Activity Assay Kit (ab65357) uses a convenient fluorescence method for detecting beta secretase activity in biological and purified samples.


    The beta secretase assay protocol uses a secretase-specific peptide conjugated to two reporter molecules, EDANS and DABCYL. In the uncleaved form, the fluorescent signal from EDANS is quenched by the physical proximity of the DABCYL moiety. Cleavage of the peptide by beta 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.

  • Notes

    β-Secretase (Beta secretase) is responsible for the proteolytic process of the amyloid precursor protein (APP). It cleaves APP at the N-terminus of the A-beta peptide sequence, between residues 671 – 672, leading to the generation and extracellular release of beta-cleaved soluble APP, and a corresponding cell-associated C-terminal fragment which is later released by gamma-secretase. It has been implicated to be an excellent target for anti-amyloid therapy for the treatment of Alzheimer’s disease

  • Platform
    Microplate reader

Properties

  • Storage instructions
    Store at -20°C. Please refer to protocols.
  • Components Identifier 100 tests
    Active ß -Secretase Red 1 x 20µl
    ß -Secretase Extraction Buffer NM 1 x 25ml
    ß -Secretase Inhibitor (in DMSO) Blue 1 x 10µl
    ß -Secretase Reaction Buffer (2X) WM 1 x 10ml
    ß -Secretase Substrate (in DMSO) Amber NM 1 x 200µl
  • Research areas
  • Relevance
    Beta secretase is a protease responsible for the proteolytic processing of the amyloid precursor protein (APP). Amyloid beta peptide is the major constituent of amyloid plaques in the brains of individuals afflicted with Alzheimers disease. This peptide is generated from the beta-amyloid precursor protein (beta APP) in a two-step process. The first step involves cleavage of the extracellular, amino-terminal domain of beta APP. Protein cleavage is performed by an aspartyl protease termed beta-secretase (BACE), of which there are two isoforms, BACE1 and 2. Beta APP cleavage by beta-secretase results in the cellular secretion of a segment of beta APP and a membrane-bound remnant. This remnant is then processed by another protease termed gamma-secretase. Gamma-secretase cleaves an intra-membrane site in the carboxyl-terminal domain of beta APP, thus generating the amyloid beta peptide.
  • Cellular localization
    Membrane; Single-pass type I membrane protein.
  • Alternative names
    • APP beta secretase
    • ASP 2
    • ASP2
    • BACE
    • BACE1
    • Beta secretase 1
    • Beta site APP cleaving enzyme 1
    • Memapsin2
    see all

Images

  • Beta Secretase Activity measured in biological fluids. Tested neat. 

  • Beta Secretase Activity measured in tissue lysates (each with protein concentration of 4 mg/mL)

  • Beta Secretase Activity measured in cell lysates (each with concentration of 2e7 cells/mL).

  • Beta-Secretase Cleavage Activity assayed using ab65357.

Protocols

References

This product has been referenced in:
  • Zamarbide M  et al. Maternal imprinting on cognition markers of wild type and transgenic Alzheimer's disease model mice. Sci Rep 8:6434 (2018). Read more (PubMed: 29691440) »
  • Blondrath K  et al. The nuclear cofactor receptor interacting protein-140 (RIP140) regulates the expression of genes involved in Aß generation. Neurobiol Aging 47:180-191 (2016). Functional Studies . Read more (PubMed: 27614112) »
See all 12 Publications for this product

Customer reviews and Q&As

1-10 of 17 Abreviews or Q&A

Abreviews
β-secretase activity in the mouse brain were measured using this kit. Solubilized membranes were extracted from brain tissues using β-secretase extraction buffer in this kit.

Ms. Ji Hye Han

Verified customer

Submitted Jun 27 2018

Answer



We recommend to use 2 – 3x volume of ice-cold extraction buffer (provided with the kit) for tissue samples and to homogenize it on ice. The composition of the extraction buffer is proprietary information but I am happy to confirm that it contains DTT, Glycerol, EDTA, & NaAc.

As Beta secretase itself is a protease, there are no proteinase inhibitors added in this kit to make sure the target enzyme itself is not inhibited.

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Answer


I contacted the laboratories. This kit has not been tested with DMSO dissolved samples. However, I am sorry it is most likely that samples in DMSO will not work well since enzymes are sensitive to DMSO. Typically >1-2% DMSO is not recommended for any activity assay or for any cell treatment. DMSO can be toxic to cells.
Successfully tested species and applications covered by the guarantee are listed on our datasheets, and this is updated as soon as we have any further information. In this case, the kit has been tested in Urine, Serum, Plasma, Tissue Extracts, Cell Lysate, Cell culture media.

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Answer



The buffer used might work but we would strongly suggest using the extraction buffer provided in the kit for the best results. The best way would be to store cell lysates at -80C if needed. The number of cells mentioned in the datasheet are based on our experience with this kit. The customer can use lower number of cells but the RFU value might be low. It is possible to optimize with less cells by using a concentrated sample lysate (that is making the lysate in less volume than recommended).

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Answer

Thank you for contacting us. This kit does allow you to measure BACE1 from cells without isolating the enzyme. We have tested this protocol and made the protocol from use with cells that were lysed using the kit's extraction buffer.

The publications this kit has been used in are listed below. They can also be found from our online datasheet. Please let me know if you have any further questions.

Jin P et al. Anti-inflammatory and anti-amyloidogenic effects of a small molecule, 2,4-bis(p-hydroxyphenyl)-2-butenal in Tg2576 Alzheimer's disease mice model. J Neuroinflammation 10:2 (2013). Functional Studies ; Mouse . PubMed: 23289709

Kindy MS et al. Deletion of the cathepsin B gene improves memory deficits in a transgenic ALZHeimer's disease mouse model expressing AßPP containing the wild-type ß-secretase site sequence. J Alzheimers Dis 29:827-40 (2012). PubMed: 22337825
Hook G et al. The cysteine protease inhibitor, E64d, reduces brain amyloid-ß and improves memory deficits in Alzheimer's disease animal models by inhibiting cathepsin B, but not BACE1, ß-secretase activity. J Alzheimers Dis 26:387-408 (2011). PubMed: 21613740
Wen L et al. VPS35 haploinsufficiency increases Alzheimer's disease neuropathology. J Cell Biol 195:765-79 (2011). PubMed: 22105352
Cui J et al. Morphine protects against intracellular amyloid toxicity by inducing estradiol release and upregulation of Hsp70. J Neurosci 31:16227-40 (2011). PubMed: 22072674

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Answer

Since this is an enzymatic assay, I would not recommend using a buffer containing EDTA.

Yes, you can add reaction buffer to the substrate. So if you are adding 18 ul of the buffer to 2 ul of the substrate, add only 32 ul of the buffer in the prior step to account for the increased volume in the next step in the protocol.

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Answer



Tips to ensure maximum beta secretase activity include:
1. Ensure assay buffer at room temperature
2. Perhaps protein in your sample is inhibiting somehow beta secretase activity. To test this, try spiking active beta secretase control into your samples and compare readings to positive control without your sample, to determine if factors are inhibiting enzymatic activity

Also, I think it would be helpful if you could send along data for review.

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Answer

wenn nach der Beratung mit ihrem Chef weitere Fragen aufkämmen können sie sich gerne nocheinmal melden.

Ich wünsche ihnen weiterhin viel Erfolg mit ihren Experimenten

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Answer

Vielen Dank für ihre Anfrage.

Für den Versuchsaufbau dieses Kits sollten in jedem Fall immer die positiv und die negativ Kontrollen sowie verschiedene Konzentrationen der Proben gemessen werden. Wenn Sie ein geeignetes Gerät verwenden in welchem die Proben nach Protokoll im Dunkeln inkubiert werden können sollte es möglich sein auch den Zeitverlauf der Reaktion zu vermessen. Das Kit liefert ein optimales Resultat nach der im Protokoll beschriebenen ein stündigen Inkubationszeit. Das ein optimaler Kurvenverlauf bei einer Messung über die Zeit erhalten wird können wir nicht garantieren.

Für die Auswertung ist es wichtig nur die Proben von gleichen Zeitpunkten bezüglich der Konzentration zu vergleichen. Für den Zeitverlauf sollten dementsprechend nur Proben der gleichen Konzentration für die Auswertung verglichen werden.


Ich hoffe diese Information ist hilfreich. Wenn Sie weitere Fragen haben zögern sie nicht sich nocheinmal bei uns zu melden.

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Answer

Thank you for contacting us with your questions about ab65357.

Do you know what the detection bandwidth is on your machine? If the bandwidth is ±20, you can use this wavelength, but if not, then unfortunatelythe difference inwavelengths will affect the reliability of the assay results.

I have looked through the publications that we are aware of in which this kit was used, and I could not find anyreference of using this kit with CSF samples. I am sorry that we do not have more information in this regard.

Please let me know if you have any further questions or if there is anything else that we can do for you, and I'll be happy to help.

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1-10 of 17 Abreviews or Q&A

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