Overview

  • Product name

    Donkey Anti-Rabbit IgG H&L (DyLight® 488)
    See all IgG secondary antibodies
  • Host species

    Donkey
  • Target species

    Rabbit
  • Specificity

    By immunoelectrophoresis and ELISA this antibody reacts specifically with Rabbit IgG and with light chains common to other Rabbit immunoglobulins. No antibody was detected against non-immunoglobulin serum proteins.
  • Tested applications

    Suitable for: IHC-P, ICC/IF, Flow Cytmore details
  • Conjugation

    DyLight® 488. Ex: 493nm, Em: 518nm

Properties

  • Form

    Liquid
  • Storage instructions

    Shipped at 4°C. Store at +4°C.
  • Storage buffer

    Preservative: 0.09% Sodium azide
    Constituents: 0.2% BSA, PBS
  • Concentration information loading...
  • Purity

    Immunogen affinity purified
  • Purification notes

    This antibody was isolated by affinity chromatography using antigen coupled to agarose beads and conjugated to DyLight® 488.
  • Clonality

    Polyclonal
  • Isotype

    IgG
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab96891 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
IHC-P 1/50 - 1/500.
ICC/IF 1/50 - 1/500.
Flow Cyt 1/50 - 1/200.

Images

  • Emission spectra of DyLight® fluorochromes available in our catalog.
    Line colors represent the approximate visible colors of the wavelength maxima.

References

This product has been referenced in:

  • Jarry AC  et al. Neuromedin U is a gut peptide that alters oral glucose tolerance by delaying gastric emptying via direct contraction of the pylorus and vagal-dependent mechanisms. FASEB J 33:5377-5388 (2019). Read more (PubMed: 30753087) »
  • Dhanani KCH  et al. Fibronectin is a stress responsive gene regulated by HSF1 in response to geldanamycin. Sci Rep 7:17617 (2017). Read more (PubMed: 29247221) »
See all 9 Publications for this product

Customer reviews and Q&As

1-7 of 7 Abreviews or Q&A

Question
Answer

Thank you for your reply.

I googled for flourocene filter, but could not find an entry. Do you maybe mean fluorescence filter?

What wavelength does the filter cover? I assume it would be in the green range(about 480 - 570 nm) since Dylight 488 is in the green range.

We have the following green fluorophores FITC, Cy2 and Chromeo 488 and Dylight 488.
However, Dylight 488 is the most photostable of these fluorophores when using lasers. Otherwise, FITC would be suitable as well, and I would suggest:

either ab98502 (donkey anti-rabbit, FITC, pre-absorbed) (https://www.abcam.com/Donkey-polyclonal-Secondary-Antibody-to-Rabbit-IgG-H-L-FITC-pre-adsorbed-ab98502.html)

or ab6717 (goat anti-rabbit, FITC) (https://www.abcam.com/Rabbit-IgG-secondary-antibody-H-L-ab6717.html)

but we have other options as well.

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

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Answer

Thank you for your email.

To answer your questions and make some comments/suggestions that hopefully will help you further:

1) The antibodies (shipped as is) should be stored aliquoted and stored at -20 or -80 degree. This would also apply to the 1/10 stock solution as well. Please make sure to avoid freeze-thaw cycles. For ab6994, a brief period of storage at +4 degree for 1-2 weeks would be fine, but for long term storage -20 or -80 degree is recommended.

2) For ab6994, you should have received 100 ul, and for ab104225 you should have received 50 ul. You can spin the vials briefly before opening and the liquid should be at the bottom of the tube and it would be visible. In thecase of these 2 antibodies, diluting them to 1/10it might be OK as they are either whole antiserum (contain other proteins) or contain lots of BSA as stabilizer.
In general, it is recommended to dilute the antibody to the working solution just before use.

A 1/100dilution would be an increase in antibody concentration compared to 1/500 (which is more dilute), as 1/100 contains more antibody than 1/500.

3) Yes, the higher temperaturewill help with the antigen retrieval.

4) Yes, this is probably fine. But in case of no signal, you might consider reducing the time to e.g. 2x 30 sec.

I'm glad to hear you are getting now signal with the Fox 3 antibody. As for the VWF antibody, would also use more primary and secondary antibody, increase the retrieval temp to 37 degrees and reduce the wash time. Yes, checking the reference could provide additional helpful information.

I wish you good luck and look forward to hear back.

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Question

The trypsin antigen retrieval did not work. I'd like to review what I did to see if there are any large errors.

Rats wre perfused in situ and brains were preserved in 4% paraformaldehyde solution overnight. We purchased the paraformaldehyde commercially pre-prepared. We then sucrose protected the brains (after speaking with an abcam rep) in a sucrose series to freeze protect them. We then froze the brains for a period of about a month.

I recieved the Fox-3 antibody from abcam as a 50ul concentrate. In reading the antibody reviews online I saw that our target antibody concentration would be between 1:500-1:2000. So I initially added 500ul to the 50 ul concentrate to have an initial 1:10 dilution (roughly). Then I took 1 ul of this working solution and diluted it with 200 ul PBS to get my 1:2000 dilution. I followed this with 1ul working solution in 100 ul PBS to get my 1:1000 dilution. Then, 2:100ul PBS to get my 1:500 dilution. I did this because an abcam representative said I should shoot for 100ul per slice.

I took my test slices, washed them in PBS for 5 minutes, then outlined them in a histology pen, and applied the primary. I spoke with an abcam rep last week who advised us not to block unless we had too much non-specific staining. I used the trypsin enzymatic retrieval kit you recommended which entained mixing the trypsin concentrate with a diluent included in the kit at a ratio 1:1. I applied 100ul of this per slice for 15 minutes at room temperature under a fume hood (not in the dark--this was not required in the literature). I then washed twice with PBS, and I applied the primary on a counter top at roomtemperatureand then stored in at 4 degrees c overnight in a dark container. After this, I rinsed the slices in PBS, and thenbathed the slices twice in PBS to get the primary off, and then applied the Dylight secondary (dilution info below) for one hour at room temp in a dark environment. I then washed once with PBS and applied the DAPI mounting medium we purchased from abcam. I coverslipped the slides and left them for about an hour at 4 degrees c in a dark slide holder. I then attempted microscopy.

Secondary info: (Donkey anti rabit Dylight 488) from its stock solution and read online that a 1:50-1:200 dilution would be optimal so I tried 1:100 which should have produced at least some fluorescence. I took the equivalent of 1ul Dylight to 99ul PBS done in the dark..

The VWF antibody was prepared the exact same way except the suggested concentrations were slightly different.
I took the 100ul concentrated antibody that abcam shipped and immediately added 900ul for a 1:10 working solution dilution. Then I did an experiment with 1:200 (5ul working solution in 95ul pbs); 1:500 (2ul working in 98 ul pbs); and 1:800 (2 ul working in 198 pbs). Secondary was applied at same conc.

Where should we go from here?

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Answer

Thank you for your email.
I am sorry to hear the antigen retrieval step did not improve your results.

I have looked throught your protocol you sent me and have the following suggestions, comments and questions:

1) You are making a 1/10 stock solution for each antibody. How do you store the antibody afterwards?

2) Also, in my understanding, diluting the entire vial of antibody - although it makes it easier for pipetting - can lead to a loss of antibody to the tube wall due to absorption andan increase in volume. Thus, increasing the primary antibody concentration might be necessary(e.g. 1/100).

3) For the enzymatic antigen retrieval, a temperatue of 37 degree would be recommendedas this is the temparature where the enzyme is most active.

Here is the link to our antigen retrieval protocol page: https://www.abcam.com/index.html?pageconfig=resource&rid=11488. In section 3, you can find information about the enzymatic retrieval.

4) In your protocol description, you mentioned: "I rinsed the slices in PBS, and then bathed the slices twice in PBS to get the primary off". How long did you bathe the slides in PBS each time? If this is done for too long, you might potentially also lose primary antibody.

5) As for the secondary antibody, it will only bind specifically if the primary antibody is present. Thus, doing the antigen retrieval at a higher temperature and using potentially more primary antibody should help with that. In addition, you might also want to try a lower dilution and longer incubation time with the seconadry antibody (e.g. 1/50 for 2 h).

6) You are using perfused frozen sections which we abbreviate IHC-FoFr.
For the VWF antibody (ab6994) this application has been tested, and it was added based on the following publication:
Fujimoto KL et al. Naive rat amnion-derived cell transplantation improved left ventricular function and reduced myocardial scar of postinfarcted heart. Cell Transplant 18:477-86 (2009). IHC-FoFr; Rat. PubMed: 19622235
It might be worthwhile checking this publication for additional protocol details or contacting the authors for the details of their technique.
In addition, a customer submittedthe following Abreview for IHC-FoFr:
https://www.abcam.com/index.html?datasheet=6994&tab=abreviews&intabreviewid=11467

As for the FOX3 antibody (ab104225) this application has not been tested yet, and we do not have information if and how well IHC-FoFr would work with this antibody. Thus, we also do not havea specific protocolestablished for this antibody. Since IHC-P was tested with ab104225, IHC-FoFr might work as well. Here is the link to the Abreview a customer submitted for IHC-P:
https://www.abcam.com/index.html?datasheet=104225&tab=abreviews&intabreviewid=25847

I have also attached a paper describing how heat-inducedantigen retrieval can be used on frozen sections.

I hope this information will be of help to you. Please let me knowif these suggestions are improving your results or not.

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Answer

Thank you for your reply.
I'm glad to hear that the secondary antibody shows fluorescence.

As for selecting another secondary antibody, could you please let me know which fluorophore or wavelength range is covered by your miscroscope? Based on that informationI can then select a different antibody for you.

Please let me know. Thank you!

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Answer

Thank you for contacting us.
I am sorry to hear that the antibodie might not be working as expected.

As we discussed over the phone the following troubleshooting tips might help in finding the cause of the problem:

1) Try antigen retrieval (enzymatic) using one of the products we discussed (ab64201, ab64220, ab64205 or ab970).

2) Check if secondary antibody works (e.g. with fluorescent plate reader or fluorescence micrscope, or with another primary Ab)

Please let me know if and how your results improving.
I wish you good luck and look forward to hear back from you.

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Answer

It has been a pleasure to help. We are here until 8:00pm eastern time so not quite as late as the university crowd. Please feel free to contact me if you have any questions.

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Question
Answer

Thank you for contacting Abcam. We have a large number of secondary antibodies with fluorescent tags. Theeasiest way to find the one that best fits your needs is from the Abcam home page. The secondary antibodies drop down, located in the yellow banner, has a listing of our DyLight conjugated secondary antibodies. You may then choose the conjugation that is best for your needs. In order to assist your search, I have chosen two products that may work for you. Both are against Rabbit IgG and will work in flow cytometry and IHC. These particular antibodies are conjugated to our DyLight fluorochromes but other fluorochromes are available. The product that I have chosen are ab96891 :https://www.abcam.com/Donkey-polyclonal-Secondary-Antibody-to-Rabbit-IgG-H-L-DyLight-488-ab96891.html and ab96883: https://www.abcam.com/Goat-polyclonal-Secondary-Antibody-to-Rabbit-IgG-H-L-DyLight-488-ab96883.html In regards to your second question. Isotype controls are essential in flow cytometry and recommended in IHC.The appropriate isotype control for this product isab27478. This product has been tested in flow cytometry and IHC-P. I have included the link to a pdf which details our recommended controls for flow cytometry. I hope this information is useful. https://www.abcam.com/ps/pdf/protocols/flow_cytometry_controls.pdf

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