Anti-N epsilon gamma glutamyl Lysine 抗体 [81D4] (ab424)

Reviews (2)Q&A (16)References (13)

製品の概要

  • 製品名
    Anti-N epsilon gamma glutamyl Lysine antibody [81D4]
  • 製品の詳細
    Mouse monoclonal [81D4] to N epsilon gamma glutamyl Lysine
  • 由来種
    Mouse
  • アプリケーション
    適用あり: IHC-P, ELISA, IHC-Frmore details
  • 免疫原

    Synthetic isopeptide.

  • 特記事項
    The ability of this antibody to fix complement is unknown. The isopeptide may be purchased from Bachem Catalog number G-1970.

製品の特性

アプリケーション

Our Abpromise guarantee covers the use of ab424 in the following tested applications.

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

アプリケーション Abreviews 特記事項
IHC-P 1/50 - 1/800.
ELISA Use at an assay dependent concentration.
IHC-Fr 1/50 - 1/800.

画像

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-N epsilon gamma glutamyl Lysine antibody [81D4] (ab424)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-N epsilon gamma glutamyl Lysine antibody [81D4] (ab424)
    ab424 staining N epsilon gamma glutamyl Lysine in skin tissue sections.
  • Immunohistochemistry (Frozen sections) - Anti-N epsilon gamma glutamyl Lysine antibody [81D4] (ab424)
    Immunohistochemistry (Frozen sections) - Anti-N epsilon gamma glutamyl Lysine antibody [81D4] (ab424)
    ab424 staining N epsilon gamma glutamyl Lysine in kidney tissue sections by Immunohistochemistry (Frozen sections). Sections A through D were stained for insoluble tissue transglutaminase, while E through H were stained for N epsilon gamma glutamyl Lysine crosslink. A, B, E, F were control kidneys; C, D, G, H were diseased kidneys showing increased staining in the expanded interstitium, the mesangium and periglomerular.

プロトコール

参考文献

This product has been referenced in:
  • Mižíková I  et al. Targeting transglutaminase 2 partially restores extracellular matrix structure but not alveolar architecture in experimental bronchopulmonary dysplasia. FEBS J N/A:N/A (2018). Read more (PubMed: 29935061) »
  • Witsch TJ  et al. Transglutaminase 2: a new player in bronchopulmonary dysplasia? Eur Respir J 44:109-21 (2014). Read more (PubMed: 24603819) »
See all 13 Publications for this product

ab424 を使用した論文を発表された方は、こちらまでお知らせください。データシートに掲載させていただきます。

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1-10 of 18 Abreviews or Q&A

Immunocytochemistry/ Immunofluorescence abreview for Anti-N epsilon gamma glutamyl Lysine antibody [81D4]

 Excellent
Abreviews
abreview image
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (Trabecular Meshwork)
Permeabilization
No
Specification
Trabecular Meshwork
Blocking step
PBS superblock blocking buffer (Thermo Scientific, Prod # 37517) as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 23°C
Fixative
Paraformaldehyde
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Abcam user community

Verified customer

投稿 Jul 21 2015

Question

It seems the IgM secondary antibody gave a good signal in at least some samples but only with overnight/4C incubation and not 2hr/RT. I run out of ab424 and I need to re-order. Can you check for me if the current lot you sell is still the same. I do not want to go over troubleshooting again now that we see at least some results, I want to use the exact same lot number. Many thanks.

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Answer

Thank you very much for keeping me updated.

I'm glad to hear that the results seem to have improved. We currently have only that lot in stock (2 vials in our US stock and 1 vial in our UK stock), so there should be consistency in results.

Please let me know if you have further questions or need anything else, and I'll be happy to help.

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Question

That would be great. I would order ab97230. You can ship at the address in my signature block below. Thank you, I appreciate it.

PS Would you mind sending along the Cell Imaging book (I have promo CELL-TBVCQ that expires Nov 16). Thank you!

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Answer

Thank you very much for your reply.

I'm sending a free of charge vial of ab97230 on the order ***, which should ship out tonight and arrive tomorrow.

I've also included the promo code for the cell imaging book.

Please keep me updated about the results with this secondary antibody, and let me know if you need anything else.

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Question

If you have the same lot, there is no point to try it I guess. We are going
to try IgM. I found ab97230 and ab98679. Which one do you recommend?

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Answer

Thank you very much for your reply.

For your assay, either of these antibodies will be suitable. I would suggest ab97230 because it is in stock and we can ship it to you overnight. Ab98679 is back-ordered and I expect it to work similarly to ab97230.

I'd be happy to send this to you free of charge if you would like. Please let me know your order information and I will set this up for you.

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Question


we did try also without antigen retrieval but we did not see any staining. I am currently using lot ***. I appreciate your offer of sending new ab. Would it be possible for me to ship you couple of slides, the same that we stained with no success, to see if you can make it work in-home? I can also send an aliquot of the ab I am using if you want to compare.

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Answer

Thank you very much for your reply.

I apologize but we won't be able to test the antibody with your samples in our lab at this time. I'm very sorry for the inconvenience. Did you check whether the mouse-on-mouse HRP polymer detection kit works with mouse IgM isotype primary antibodies? If you'd like to send me the catalog number of the kit you're using, I'll be happy to look into that for you.

We only have the same lot in stock that you're currently using, but we can still send a replacement vial to see if the results improve. If you'd like to try this, please send me your original order or PO number and I'll arrange this for you.

I am sorry that I don't have more to offer at this time, but please let me know if you have any questions or need anything else and I'll be happy to help.

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Question

I contacted you a few weeks ago in regard to crosslinks antibody (ab424) that we used on formalin-fixed paraffin embedded aortic sections. We run a few tests changing the retrevial time, temperature of retrivial and reagent (citrate vs trypsin); we tried 2 hour incubation at RT and overnight at 4C. Unfortunately none of this worked: the immunostaining is not there even if we use 1:50 dilution antibody (see representative pic below). Do you have any suggestions? I am not sure how to procede to get good results. Please note this ab was suggested to me from one of the authors in attached paper that looked at crosslinks and it shows it is expressed in mouse aortas under basal conditions.

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Answer

Thank you very much for keeping me updated about the results.

I'm sorry to hear that altering the protocol did not help improve the results. Have you also tried staining sections without doing antigen retrieval at all? I apologize that we don't have an in-house IHC-P protocol for this antibody, as it has only been tested in this kind of tissue staining according to the literature. Are you certain that your secondary antibody or detection system is reactive with mouse IgM isotype? An anti-IgG secondary will probably react with the conserved light chains of the IgM molecules, but this wouldn't be as strong as an anti-IgM staining.

What lot number are you using right now? We have lot a new lot in stock, and I'd be happy to send you a free of charge vial to see if the results are different. If you'd like to try this, please let me know your original order or PO number and I'll be happy to send this to you.

I look forward to hearing from you. Please let me know if you have any questions or need anything else and I'll be happy to help.

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Question

I gathered a few more info on the methods used with ab424:

1: antigen retrievial was done using a decloaking machine with Rodent Decloaking Agent. It was turned on 90C for 35 min with a cooldown of 85C for 10 min

2: blocking done with Peroxidase 1 for 10 min and Rodent Block M (blocker for rodents anti-mouse igg) for 30 min

3: the ab was incubated for 2 hrs at RT

4: detection was done with Mouse on Mouse HRP polymer for 30 min

5: Primary ab was diluted with DaVinci Diluent. I used 1:50 dilution for ab424 but saw a very week stain (as you saw in pics I sent previously). Secondary ab was Ready to Use

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Answer

Thank youvery much for your reply and for sending these additional details.

I have a couple of suggestions that might help improve the results. It may be useful to decrease the antigen retrieval time to 15-20 minutes, in order to reduce the reversal of cross-linkages. Do you know if the rodent decloaking agent is similar to citrate buffer at pH 6, which has previously worked in antigen retrieval with this antibody? I'd also recommend incubating the antibody overnight at 4C instead of 2 hours at room temperature.

If these alterationsare not helpful in improving the results with this antibody, I'dbe happy to send a free of charge replacement antibody orissue a credit or refund. Please keep me updated about any further results, and let me know if you have any questions or there is anything else that we can do for you.

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Question

Ifinally got your e-mail. Thanks for looking into this issue. We did antigen retrieval with an unmasking reagent in decloaking chamber at 90C. I know that this cross-links once formed are very stable so even rush conditions should not break them however I was surprised to see a very weak signal with 1:50 antibody dilution (the highest we can use). Below some examples of one control aorta section (upper) and one treated animal (lower) where we expected to see a strong signal but we saw only a slight increase even with such highly concentrated antibody.

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Answer

Thank you very much for your reply, and for your patience.

Thank you also for sending the images of your staining results. I've reviewed some of the literature in which this antibody was used in IHC-P, and there aren't many more details about the protocols used. Have you tried increasing the incubation time, such as overnight at 4C if you're currently incubating for 1 hour at room temperature? What kind of blocking solution is being used? I'd recommend blocking with 10% serum for up to an hour at room temperature. What kind of secondary antibody and detection system is being used? Could an amplification be done (avidin-biotin complex, etc)?

If none of these ideas are useful, then I'd be happy to send a replacement vial or issue a credit or refund. Please keep me updated with any new results, and let me know if you have any questions or if there is anything else that we can do for you.

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Question

Was antigen retrieval used on formalin-fixed paraffin-embedded tissue?
This is a crosslinked epitope and antigen retrieval reverses crosslinks, so it may be an unsuitable step.

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Answer

Thank you very much for your call last week and for your patience while I've looked into your enquiry.

We have not tested this antibody in our own labs in IHC on formalin-fixed paraffin-embedded tissue, but it has been testedin IHC-P according tothe literature. One publication by Watts et al indicated that they used an Unmasking Reagent from Vector-

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2682564/

In another article by Tovar-Vidales et al, 10 mM citrate buffer pH 6 was used for antigen retrieval-

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2648869/

I haven't found any references showing that antigen-retrieval was not used, so we don't know if the antibody works better without this step. If you'd like to send some more details about your protocol, I'll be happy to review it and see if there are any suggestions to improve the results. We do fully guarantee this antibody to work in IHC-P, so we can send a replacement or offer a credit or refund if the results are still not satisfactory.

I look forward to hearing from you. If you have further questions or need anything else, please let me know and I'll be happy to help.

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Question

May I check with you, for the N epsilon gamma glutamyl Lysine antibody [153-81D4] (ab424), the species reactivity is not stated in the antibody datasheet, so could I just confirm with you that I will be able to use this antibody for human cells?

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Answer

I can confirm that this antibody will detect N epsilon gamma glutamyl Lysine also if part of a human protein. In the publication PubMed ID 18235007 for example, researchers have used this antibody successfully in human paraffin embedded tissue slides. We actually have not stated species reactivity on the datasheet, as N epsilon gamma glutamyl Lysine is a very small epitope and its recognition should therefore not be dependent on the species.

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