Anti-Firefly Luciferase antibody [Luci17] (ab16466)

Reviews (3)Q&A (10)References (13)

Overview

  • Product name

    Anti-Firefly Luciferase antibody [Luci17]
    See all Firefly Luciferase primary antibodies

  • Description

    Mouse monoclonal [Luci17] to Firefly Luciferase

  • Host species

    Mouse

  • Tested applications

    Suitable for: Flow Cyt, WB, IHC-Frmore details

  • Immunogen

    Hybridoma produced by the fusion of spleenocytes from mice immunized with luciferase protein isolated from Photinus pyralis.

  • Positive control

    • WB: Purified luciferase protein.

  • General notes

Properties

  • Form

    Liquid

  • Storage instructions

    Shipped at 4°C. Upon delivery aliquot and store at -20°C. Avoid freeze / thaw cycles.

  • Storage buffer

    Preservative: 0.08% Sodium Azide
    Constituents: PBS, pH 7.2
  • Concentration information loading...

  • Purity

    Protein A/G purified

  • Clonality

    Monoclonal

  • Clone number

    Luci17

  • Isotype

    IgG1

  • Light chain type

    unknown

  • Research areas

Applications

Our Abpromise guarantee covers the use of ab16466 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
Flow Cyt Use at an assay dependent concentration. PubMed: 18612415

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.
WB Use at an assay dependent concentration.
IHC-Fr Use at an assay dependent concentration.

Target

  • Relevance

    Luciferase from the firefly has become one of the more widely used reporter proteins for the study of gene expression. Luciferase catalyzes a bioluminescent reaction which requires the substrate luciferin as well as Mg2+ and ATP. Mixing these reagents with the cell extract containing luciferase, results in a flash of light that decays rapidly. This light can be detected by a luminometer. The total light emission is proportional to the luciferase activity of the sample.

  • Cellular localization

    Peroxisome

  • Alternative names

    • ec 1.13.12.7 antibody
    • Firefly antibody
    • Luciferase antibody
    • Luciferin 4 monooxygenase antibody

Images

  • Western blot - Anti-Firefly Luciferase antibody [Luci17] (ab16466)
    Western blot - Anti-Firefly Luciferase antibody [Luci17] (ab16466)
    All lanes : Anti-Firefly Luciferase antibody [Luci17] (ab16466) at 1 µg/ml

    Lane 1 : Non-transfected 293 whole cell lysate
    Lane 2 : Firefly Luciferase transfected 293 whole cell lysate
    Lane 3 : HeLa whole cell lysate
    Lane 4 : NIH3T3 whole cell lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed (ab216772) at 1/10000 dilution

    Performed under reducing conditions.

    Observed band size: 65 kDa
    why is the actual band size different from the predicted?



     

    Lanes 1 - 4: Merged signal (red and green). Green – ab16466 observed at 65 kDa. Red - loading control, ab181602, observed at 37 kDa.

     This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using Licor blocking buffer before being incubated with ab16466 and ab181602 (loading control) overnight at 4°C. Antibody binding was detected using Goat anti-Mouse IgG H&L (IRDye® 800CW) preabsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed (ab216777) at a 1:10000 dilution for 1hr at room temperature and then imaged.

  • Flow Cytometry - Anti-Firefly Luciferase antibody [Luci17] (ab16466)
    Flow Cytometry - Anti-Firefly Luciferase antibody [Luci17] (ab16466)Image from Pellegatti P et al., PLoS One. 2008 Jul 9;3(7):e2599 Fig 5.; doi:10.1371/journal.pone.0002599; July 9, 2008, PLoS ONE 3(7): e2599.
    Flow cytometric analysis of HEK293 cells expressing Firefly Luciferase, staining Firefly Luciferase with ab16466.

    Cells were fixed with paraformaldehyde at room temperature for 20 min, rinsed twice with permeabilization buffer (PBS, 1% FBS, 0.1% saponin), and incubated with primary antibody. A FITC-conjugated anti mouse IgG was used as the secondary antibody.

References

This product has been referenced in:

  • Afzal S  et al. Rat Glioma Cell-Based Functional Characterization of Anti-Stress and Protein Deaggregation Activities in the Marine Carotenoids, Astaxanthin and Fucoxanthin. Mar Drugs 17:N/A (2019). Read more (PubMed: 30909572) »
  • Forootan SS  et al. Real-time in vivo imaging reveals localised Nrf2 stress responses associated with direct and metabolism-dependent drug toxicity. Sci Rep 7:16084 (2017). Read more (PubMed: 29167567) »
See all 13 Publications for this product

Publishing research using ab16466? Please let us know so that we can cite the reference in this datasheet.

Customer reviews and Q&As

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1-10 of 10 Q&A

Question

Primary antibody dilutions are not specified either in the website or in the paper that used this antibody for flow cytometry and are cited in the website.

Could you give me this information so I can have a starting point?

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Answer

The flow cytometry image shown on ab16466's datasheet is from: Pellegatti P et al. Increased level of extracellular ATP at tumor sites: in vivo imaging with plasma membrane luciferase. PLoS ONE 3:e2599 (2008) as stated in the figure legend.

From the materials section of this paper, it states that cells were incubated with ab16466 (8–10 µg/5×10^5 cells.) I would therefore use this as a starting point for your experiment.

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Question

We are using pGL3 Luciferase Reporter Vector. It has a modified coding region for firefly (Photinus pyralis) luciferase that has been optimized for monitoring transcriptional activity in transfected eukaryotic cells.


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Answer

Thank you for the clarification. EIther one of these antibodies should work well with your sequence. I personally would select ab16466 because it is a purified antibody rather than an IgG fraction, and therefore will have less chance of non-specific binding. I hope this information is helpful to you. Please let me know if I can be of further assistance.

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Question

I have purchased an antibody (ab16466) which is 1mg/ml and i have to make 400µg/ml, can you tell me what buffer do i need to dilute it.

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Answer

Thank you for your enquiry.


Usually the buffer to use for dilutionis the same as your blocking buffer. There are some common buffers that are used which are1-3% BSA buffer, secondary antibody host species serum, Blockase, or even just PBST only.


Please check the protocols on our website for more details.
https://www.abcam.com/index.html?pageconfig=popular_protocols


I hope this information will be helpful. If there is anything else that I can help you with please do not hesitate to contact me.


Have a nice day!

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Question

Good Afternoon abcam Technical Service,
I am looking to find out what the sensitivity of your two monoclonal antibodies: Anti-firefly Luciferase antibody [Luci 21 1-107] (ab7358) and Anti-Firefly Luciferase antibody [Luci17] (ab16466) is?

Thank you in advance for your help.

Sincerely,

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Answer

Thank you very much for contacting us with your question, and for your patience while I have been in touch with the lab regarding these antibodies.

Unfortunately, the sensitivities of these two antibodies has not been determined at this time.

I am sorry that I do not have further information for you, but if there is anything else that we can do for you please let me know and I'll be happy to help.

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Question

What is the sensitivity of this antibody in a western blot against a recombinant protein in western blot?

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Answer

Thank you for contacting Abcam

We have a western blot image, which is attached showing the antibody with recombinant luciferase. We have not done further testing to see what is either smallest concentration of antibody that is needed to recognize a know concentration of the protein.

If there is anything else I can help you with, please let me know.

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Question

Thanks for your quick reply. Now i know that you have ab16466 Mouse monoclonal [Luci17] . I just wanted to know if you have any-other anti-firefly antibody raised not in mouse.

So basically i want to to do is pull-downs with Mouse monoclonal [Luci17] and do a western with anti-firefly raised either in goat or rabbit (if possible with HRP for ECL but not required).

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Answer

I hope youmanaged to open the attachment file I sent previously. The image contains the list of non-conjugated HRPanti-Firefly antibodies we have in our catalogue, including goat polyclonal, chicken polyclonal, and rabbit polyclonal antibodies. These antibodies can be used in WB application, so I am sure you will be able to find one suitable for your experiment.



Our current stock status for these antibodies are as below:



ab498 Goat polyclonal to Firefly Luciferase (1 local stock)

ab81823 Goat polyclonal to Firefly Luciferase (1 local stock)

ab99944 Rabbit polyclonal to Firefly Luciferase (8 overseas stock, lead time 1 week)

ab18595 Chicken polyclonal to firefly Luciferase (no stock, lead time 1-2 weeks from order)



I hope the stock information will help you decide on which product to use for your experiment.



Good luck with your researchand have a nice day!

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Question

I need to find out if you have anti-firefly antibody for IP and do a western subsequently. If yes can you send me a protocol for IP with anti-firefly antibody.

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Answer

Thank you for your enquiry.



We do have a few Firefly antibodies in our catalogue (see attached file)but unfortunately, none of them have been tested in IP application. These products however can be used in WBand might also work in IP.For your information, one of our best selling antibodies is ab16466 Mouse monoclonal [Luci17] to Firefly Luciferase.



Please rest assured that all tested applications are specified on Abcam product datasheets, and these are updated as soon as any new information is brought to our attention. Although IP is not stated on the datasheet, this does not mean that the antibody will not work, just that we can not guarantee that you will get good results. Please rest assured that if we have tested it in IP and found that it does not work, the application will be stated on the datasheet as not suitable for the product.



Regarding ourrecommended IP protocol, please see the following link for more information.



https://www.abcam.com/index.html?pageconfig=resource&rid=11385



I hope this information will still be helpful.If there is anything else that I can help you with please do not hesitate to contact me.



Have a nice day!

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Question

Dear technical team,

Our customer would like to know ab16466hasreactivity to monkey sample.
https://www.abcam.com/Firefly-Luciferase-antibody-Luci17-ab16466.html
Check this please.

Best regards,

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Answer

Thank you for your enquiry and your interest in our products.

Luciferase from the firefly and the luciferase genes can be synthesized and inserted into organisms or transfected into cells. Normally, monkeys do not express luciferase unless the samples contain transfected cells.

If you need any further assistance in the future, please do not hesitate to contact me.

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Question

Hi, I have a question on your antibody product, Ab16466, a monoclonal antibody for firefly luciferase. You mention that it is " a Hybridoma produced by the fusion of spleenocytes from mice immunized with luciferase protein isolated from Photinus pyralis." Can I confirm that this antibody was produced by immunization using the whole, native protein, as opposed to a shorter peptide epitope? Can I also ask if you've identified the epitope in firefly luciferase that the antibody reacts with? Also, have you ever tested to see if the antibody inhibits the enzymatic activity of firefly luciferase?

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Answer

Thank you for contacting Abcam.

This product was created using the whole purified protein and an immunogen. The fusion of spleenocytes is an antibody production technique for creating monoclonals.

The epitope for this antibody has not yet been mapped.

To our knowledge this product has not been tested for inhibition of enzymatic activity.

I hope this information is helpful. Please let me know if you have any questions.

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Question

I need firefly luciferase antibody for WB. I have reporter gene Renilla Luciferase and Firefly luciferase both express in vitro (Rabbit Recticalum Lysate). As Firefly luciferase expression is quite low to be detected well using Luciferase activity assay, I need firefly luciferase antibody to confirm the its expression. I wonder the sensitive of FF luciferase antibody compared to luminescent assay. I also want to know the specificity of FF Luciferase antibody: Does it bind to Renilla Luciferase as well and how about the background in rabbit recticalum lysate? Finally, we have secondary antibody for anti-rabbit and anti-mouse. And It will be best to do IP pull down. What is the best choice for me?

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Answer

We have no firefly luciferase antibody tested in IP which is mouse or rabbit origin. Only ab498- Goat polyclonal to firefly Luciferase Other Firefly Luciferase antibodies tested in WB: 1. ab16466 Firefly Luciferase antibody [Luci17] Mouse monoclonal. Tested applications: Flow Cyt, WB, IHC-Fr 2. ab7358 Firefly Luciferase antibody [Luci 21 1-107].Mouse monoclonal. Tested applications:ICC, WB 3. ab64564 Firefly Luciferase antibody [3i1]. Mouse monoclonal. Tested applications: ICC, WB, ELISA None of the antibodies have been tested for cross-reactivity with Renilla-luciferin. I compared the sequences for Luciferin 4-monooxygenase OS=Photinus pyralis (uniProt P08659) and Renilla-luciferin 2-monooxygenase OS=Renilla reniformis (uniProt P27652) and there is no homology. If I used the wrong luciferase protein sequence for Renilla, please let me know. The background in rabbit recticalum lysate: we have no information regarding that. I would say that the choice is one of the three mouse monoclonals but keep in mind that they might not be suitable for IP as we have not tested this application. Two of the mouse monoclonals have references: ab16466, ab7358. I would recommend taking a look at the references to see if there is any information which will help you with your experiment.

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