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    cathepsin-b-activity-assay-kit-fluorometric-ab65300.pdf

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Cathepsin B Activity Assay Kit (Fluorometric) (ab65300)

  • Datasheet
  • SDS
  • Protocol Booklet
Submit a review Q&A (12)References (51)

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Functional studies - Cathespin B Activity Assay Kit
  • Functional assays: Cathepsin B Activity Assay Kit (Fluorometric) (ab65300)

Key features and details

  • Assay type: Quantitative
  • Detection method: Fluorescent
  • Platform: Microplate reader
  • Assay time: 2 hr
  • Sample type: Cell Lysate, Tissue Extracts

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Overview

  • Product name

    Cathepsin B Activity Assay Kit (Fluorometric)
    See all Cathepsin B kits
  • Detection method

    Fluorescent
  • Sample type

    Tissue Extracts, Cell Lysate
  • Assay type

    Quantitative
  • Assay time

    2h 00m
  • Product overview

    Cathepsin B Activity Assay kit (Fluorometric) (ab65300) is a fluorescence-based assay that utilizes the preferred cathepsin-B substrate sequence RR labeled with AFC (amino-4-trifluoromethyl coumarin).


    Cell lysates or other samples that contain cathepsin-B will cleave the synthetic substrate RR-AFC to release free AFC. The released AFC can easily be quantified using a fluorometer or fluorescence plate reader at Ex/Em 400/505 nm.


    The Cathepsin B assay protocol is simple, straightforward, and can be adapted to 96-well plate assays. Assay conditions have been optimized to obtain the maximal activity.


    Cathepsin B assay protocol summary:
    - add samples to wells
    - add reaction mix and incubate for 1-2 hr at 37ºC
    - analyze with microplate reader

  • Notes

    This product is manufactured by BioVision, an Abcam company and was previously called K140 Cathepsin B Activity Fluorometric Assay Kit. K140-100 is the same size as the 100 test size of ab65300.

  • Platform

    Microplate reader

Properties

  • Storage instructions

    Store at -20°C. Please refer to protocols.
  • Components Identifier 100 tests
    CB Cell Lysis Buffer WM 1 x 25ml
    CB Inhibitor (1mM) Red 1 x 20µl
    CB Reaction Buffer NM 1 x 5ml
    Substrate Ac-RR-AFC (10mM) Brown 1 x 200µl
  • Research areas

    • Kits/ Lysates/ Other
    • Kits
    • Apoptosis Kits
    • Cathepsin Assay Kits
    • Neuroscience
    • Processes
  • Relevance

    Cathepsin B is a normal lysosomal proteinase that is expressed in all cells. It is a cysteine endoproteinase structurally and functionally related to the papain family of proteases. Cathepsin B is synthesized as an inactive pro enzyme which, by removal of a 62 amino acid propeptide, is activated to the single chain form of 31 kDa or the two chain form (25 and 5 kDa subunits). Matured Cathepsin B is normally localized in the lysosomes where it functions in protein turnover. Cathepsin B is also shown capable of degrading extracellular matrix proteins at acidic and neutral pH. Endogenous inhibitors of cysteine proteases, the cystatins and stefins, may play a major role in regulating Cathepsin B activity. In tumor cells and in cells exposed to mitogens, cathepsin B displays altered cellular trafficking resulting in the secretion of the 43 kDa precursor. Elevated levels of Cathepsin B correlate with malignancy suggesting that this enzyme may be a useful prognostic marker for several types of human cancers. Cathepsin B has also been implicated in the pathogenesis of rhematoid arthritis, muscular dystrophy and tumour metastasis. It is also known as amyloid precursor protein secretase and is involved in the proteolytic processing of amyloid precursor protein (APP).
  • Cellular localization

    Lysosome. Melanosome. Note=Identified by mass spectrometry in melanosome fractions from stage I to stage IV.
  • Alternative names

    • Amyloid precursor protein secretase
    • APP secretase
    • APPS
    • Cathepsin B1
    • CathepsinB
    • CPSB
    • CTSB
    • Preprocathepsin B
    see all

Associated products

  • Related Products

    • Z-LE(OMe)VD(OMe)-FMK, derivative of Z-LEVD-FMK (ab120489)
    • Z-Phe-Phe-FMK, Cathepsin B and L inhibitor (ab141386)
    • ALLM, calpain inhibitor (ab141446)
    • Q-LEHD-OPh, caspase-9 inhibitor (ab142039)
    • AFC (7-Amino-4-trifluoromethylcoumarin), Fluorescent marker (ab145587)

Images

  • Functional studies - Cathespin B Activity Assay Kit
    Functional studies - Cathespin B Activity Assay KitBong G.J., PLoS One 8(10), Fig 5F. doi:10.1371/journal.pone.0076466 Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

    Enzyme activity of Cathepsin B and Cathepsin D was determined using Cathepsin B/D activity assay kit (ab65300 and ab65302). Neuro2a cell lysates were centrifuged at 10,000 xg for 10 minutes at 4ºC and supernatant was used for the assay. Cathepsin activity was performed at the 24 and 48 hours time points by cleavage of the fluorescence peptide substrate [DnP-DR-MCA, GKPILFFRLK(DnP)-DR substrate peptide labeled with MCA]. Data represent the mean ± SD (*p<0.05, **p<0.001).

  • Functional assays: Cathepsin B Activity Assay Kit (Fluorometric) (ab65300)
    Functional assays: Cathepsin B Activity Assay Kit (Fluorometric) (ab65300)

    Cathepsin B activity in HL60 cell lysates (2.5x10e6 cells) with the addition of inhibitor. 

Protocols

  • Protocol Booklet

Click here to view the general protocols

Datasheets and documents

  • SDS download

  • Datasheet download

    Download

References (51)

Publishing research using ab65300? Please let us know so that we can cite the reference in this datasheet.

ab65300 has been referenced in 51 publications.

  • Berning L  et al. Prodigiosin Sensitizes Sensitive and Resistant Urothelial Carcinoma Cells to Cisplatin Treatment. Molecules 26:N/A (2021). PubMed: 33673611
  • Wu Y  et al. The effects of Cstb duplication on APP/amyloid-ß pathology and cathepsin B activity in a mouse model. PLoS One 16:e0242236 (2021). PubMed: 34292972
  • Hansen L  et al. Expression of Cathepsins B, D, and G in Extracranial Arterio-Venous Malformation. Front Surg 8:676871 (2021). PubMed: 34409065
  • Zeng B  et al. Isoimperatorin (ISO) reduces melanin content in keratinocytes via miR-3619/CSTB and miR-3619/CSTD axes. Biosci Biotechnol Biochem 84:1436-1443 (2020). PubMed: 32299303
  • Wu Y  et al. Regulation of endo-lysosomal pathway and autophagic flux by broad-spectrum antipathogen inhibitor ABMA. FEBS J 287:3184-3199 (2020). PubMed: 31901207
View all Publications for this product

Customer reviews and Q&As

Show All Reviews Q&A
Submit a review Submit a question

1-10 of 12 Abreviews or Q&A

Question

Can I use a plasma sample with this assay kit?

Read More

Abcam community

Verified customer

Asked on Nov 04 2011

Answer

Thank you for your enquiry. It may be possible to use plasma samples with this kit plasma. To date, however, we have no data testing plasma samples with this kit. If you do decide to use ab65300 with plasma samples I'd encourage you to submit an Abreview to share your data with other researchers. For more information on our Abreview customer response system please see this link at www.abcam.com/reviews Please let me know if you have any further questions.

Read More

Abcam Scientific Support

Answered on Nov 04 2011

Question

Can you tell me the components of the lysis or the reaction buffer so that I can troubleshoot?

Read More

Abcam community

Verified customer

Asked on Apr 21 2014

Answer

I'm sorry we cannot share the composition of the lysis or the reaction buffer due to proprietary reasons. However, the lysis buffer contains Tween 20 which might not always work well with all enzyme-based assays.

Read More

Jeremy Kasanov

Abcam Scientific Support

Answered on Apr 21 2014

Question



I am trying to determine if this kit is sufficient for our needs but cannot find the sensitivity range listed anywhere. Is there any way I can determine this before purchasing the product.

Read More

Abcam community

Verified customer

Asked on Apr 23 2013

Answer

Thank you for contacting us.

The sensitivity of these cathepsins is: 10 – 100 ng/assay.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Read More

Abcam Scientific Support

Answered on Apr 23 2013

Question

both kits: for use with tissue samples - any specific tips and instructions?
any substances to avoid (cathepsin can be instable at a certain pH)

Read More

Abcam community

Verified customer

Asked on Mar 21 2013

Answer


The lab let me know that they do not have any additional tips for tissue processing other than the ones we mention on the FAQ site.


12. How should tissue be prepared for the kits?

Samples should be completely homogenized, preferably using a Dounce homogenizer. Some substances can interfere with the assays and should be avoided in sample preparation. Please see individual kit datasheets for more specific information on which substances may affect the specific kits, but in general, substances such as EDTA (> 0.5mM), ascorbic acid (> 0.2%), sodium azide (> 0.2%), NP-40 and Tween-20 (>1%) are known to interfere with these assays and should be avoided in sample preparation. Moreover, we recommend deproteinating the sample prior analysis using a https://www.abcam.com/10kD-Spin-Column-ab93349.html.


Based on literature the optimal pH range of cathepsin B varies between 4 - 6. Therefore they would avoid anything above pH ˜7.0.

Read More

Abcam Scientific Support

Answered on Mar 21 2013

Question

Do you offer free AFC for generating a standard curve?

Read More

Abcam community

Verified customer

Asked on Feb 06 2013

Answer

Free AFC is available as product ID ab129872.

Read More

Abcam Scientific Support

Answered on Feb 06 2013

Question

Hello,

I am interested in using this kit. What are the cross species capacities?
Will it work for rat or mouse Cathepsin B?

Thanks,

Read More

Abcam community

Verified customer

Asked on May 24 2012

Answer

Thank you for your enquiry.

I can confirm this ab65300 Cathepsin B Activity Assay Kit (Fluorometric) is compatible with all mammalian samples, so it can be used with mice and rat samples. Here are a few citations which I hope may be beneficial:

K140 :: Cathepsin B Activity Assay Kit
Ha S-D et al (2008) J. Immunol. 181: 690-697.
Irani, D. N. et al Cleavage of Cystatin C in the Cerebrospinal Fluid of Patients with Multiple Sclerosis Ann Neurol Feb 2006
Schmidt, S. et. al. Kindlin-3–mediated signaling from multiple integrin classes is required for osteoclast-mediated bone resorption. J. Cell Biol., Mar 2011; 192: 883 - 897.

Hope this information has been helpful for you. If you have any further questions, please do not hesitate to contact us.

Read More

Abcam Scientific Support

Answered on May 24 2012

Question

I order the cathepsin B and cathepsin D assay kits from Abcam. In the kit there are a cell lysis buffer without no identification about the content. I wanna use the same samples to measure both activities. Could I choose one cell lysis buffer or should I do it separetely? The cathepsin B and D lysis buffer are different?

Read More

Abcam community

Verified customer

Asked on Feb 28 2012

Answer

Thank you for contacting us.

I have had the chance to speak with the lab regarding your question. Each buffer is of different composition and they cannot be used interchangeably.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Read More

Abcam Scientific Support

Answered on Feb 28 2012

Question

Hello,

I am interested in possibly ordering the Cathepsin B Activity kit. However, when I looked at your protocol it is written to detect cathepsin B activity is cell lysates. I am wondering if it can also be used with plasma? If so, what concentration would be used?

Thank you and I look forward to a response,

Read More

Abcam community

Verified customer

Asked on Feb 03 2012

Answer

Thank you for contacting us and your interest in our products.

The Cathepsin B Activity Fluorometric Assay Kit (ab65300) has not currently been tested with plasma samples.However, as long as theplasma samples contain Cathepsin B within the detection limit of theassay10 – 100 ng/assay I see no reason why the kit should not be compatible with these samples. You wouldneed to optimise the kit for this use.

If you do decide to useab65300 with plasma samples I'd encourage you to submit an Abreview to share your data with other researchers. For more information on our Abreview customer response system please follow the link below:

https://www.abcam.com/reviews

I hope this information has been of help. If you require any further information please do not hesitate to contact us again.

Read More

Abcam Scientific Support

Answered on Feb 03 2012

Question

Signal increased after adding cathepsin B inhibitor.

Read More

Abcam community

Verified customer

Asked on Oct 10 2011

Answer

According to our records, ab65300 was proving difficult to use and we were in contact in order to help resolve the issue. Looking at our correspondence, it appears that we are awaiting more details in order to help us better understand the difficulties experienced. If the requested information has already been sent, it appears that it did not reach our Scientific Support team and we apologize for this inconvenience. In this case we would like to ask for the information again so that we can reach a resolution. If the issue has already been settled, please let us know so that we can be assured that the problem has been solved to your satisfaction and update our records. We wish you the best of luck with your research and look forward to a reply.

Read More

Abcam Scientific Support

Answered on Oct 10 2011

Question

Signal increased after adding cathepsin B inhibitor.

Read More

Abcam community

Verified customer

Asked on Sep 27 2011

Answer

Thanks for your call today and for letting us know about the problem with ab65300. As we discussed, please send your data and I will forward it on to the lab to see if there is any explanation or suggestion for your results. Could you also tell me what the cells were treated with? I look forward to hearing from you and resolving this promptly. Please let me know if you have any questions or if there is anything else we can do.

Read More

Abcam Scientific Support

Answered on Sep 27 2011

1-10 of 12 Abreviews or Q&A

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