Goat Anti-Rat IgG H&L (HRP) preadsorbed (ab98425)

Submit a review Q&A (3)

Overview

  • Product name

    Goat Anti-Rat IgG H&L (HRP) preadsorbed
    See all IgG secondary antibodies

  • Description

    Goat polyclonal Secondary Antibody to Rat IgG - H&L (HRP), pre-adsorbed

  • Host species

    Goat

  • Target species

    Rat

  • Tested applications

    Suitable for: IHC-P, ELISA, WB, ICCmore details

  • Minimal
    cross-reactivity


    Chicken, Cow, Goat, Horse, Human, Mouse, Rabbit, Sheep more details

  • Conjugation

    HRP

Properties

  • Form

    Liquid

  • Storage instructions

    Shipped at 4°C. Store at +4°C.

  • Storage buffer

    Preservative: 0.1% CMIT/MIT 3:1
    Constituents: PBS, 0.2% BSA
  • 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 Horse Radish Peroxidase.

  • Clonality

    Polyclonal

  • Isotype

    IgG

  • Research areas

Applications

Our Abpromise guarantee covers the use of ab98425 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 1/200 - 1/5000.
ELISA 1/10000 - 1/100000. (as Primary).
WB 1/5000 - 1/50000. Colorimetric: 1/5000 - 1/30000; Chemiluminescent: 1/10000 - 1/50000.
ICC Use at an assay dependent dilution.

References

ab98425 has not yet been referenced specifically in any publications.

Publishing research using ab98425? Please let us know so that we can cite the reference in this datasheet.

Customer reviews and Q&As

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1-3 of 3 Abreviews or Q&A

Question

Thank you for your support.
The RAG-specimen were even longer fixed.
I have no possibility to obtain other fixed organs, because they are the appropriate negative control and belong to a very special experiment that cannot be repeated.
I don't know if you ever did 2µm-paraffin-sections of heavily infected spleen ( the next step would be brain ), non-infected would be less difficult.
I never managed less than 4µm with the infected ones because of the tissue changes during infection ... and all the slides should have the same µm.
I try once more with 10% blocking and a fc-block prior second antibody.
Best regards

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Answer

Thank you for your email and trying the protocol tips.

Please let me know if the the results do not improve. I will be happy to issue a credit note in case the antibody does not work as stated on the datasheet.

In case the results improve, we would also be happy to hear about it.

I wish you good luck and a successful week.

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Question

I once again have an inconsistency with one of your products.
We ordered from you a preabsorbed Antibody, goat-anti-rat HRP-conjugated last week, since the last one from september 2011 goat-anti-rat HRP (ab97057) led to positive signals without any primary rat antibody.
Now we have at least a clean RAG-mouse spleen with 1:500 dilution of the new antibody, but the wildtype spleen still gives positive stainings even with 1:1000 dilution.
It is the Goat polyclonal Secondary Antibody to Rat IgG - H&L (HRP), pre-adsorbed, ab98425. I made some microscopical pictures for you and added it as a .pdf. Maybe you have an explanation for me, why it is so difficult to establish
1) Abcam product code ab98425
2) Abcam product batch number
3) Description of the problem
Positive Signals without using primary antibody ( planned: diverse rat-anti-mouse against leukocyte surface antigenes )
4) Sample preparation:
Species mouse spleen tissue, C57/Bl6 and C57/Bl6 Rag-/-
Type of sample: Fresh frozen sections, perfusion fixed frozen sections, PFA/formalin fixed paraffin embedded sections, cells in culture, other:
Sample preparation 4%PFA fixed paraffin embedded sections, 4 to 6 µm
Positive control just establishing in this lab, cd3 should serve as a reliable control
Negative control just establishing in this lab, second antibody only should serve, otherwise a suitable isotype control from fixed spleen cells would be tested
5) Fixation step
Yes/No yes
If yes: Fixative agent and concentration 4%PFA in PBS- pH7,3
Fixation time 4 hours, then changed with fresh fixation buffer for 2 days
Fixation temperature 4°C
Manual embedding after manual, extensive dehydrating procedure
6) Antigen retrieval method citric acid method, microwave 1min, full power
7) Permeabilization method: none
Did you do a permeabilization step (details please) or add permeabilizing agent in any dilution buffers? none
Permeabilizing agent and concentration: none
8) Blocking agent (eg BSA, serum.):
Concentration goat normal serum 5%, heat inactivated 56°C for 30 minutes in PBS pH7,3
Blocking time 60 min
Blocking temperature RT
9) Endogenous peroxidases blocked? Yes, 5%, 30 minutes
Endogenous biotins blocked? No, not recommended
10) Primary antibody (If more than one was used, describe in "additional notes") :
Concentration or dilution rat antibodies from santa cruz biotechnology, against CD3, CD4, CD8a, Ly6G, F4/80 paraffin suitable,
not done yet since the second antibody alone gives positive signals
Diluent buffer see blocking solution
Incubation time would be 45 minutes, RT
11) Secondary antibody:
Species: goat
Reacts against: rat
Concentration or dilution 1:200, 1:500
Diluent buffer see blocking solution
Incubation time 30 minutes, RT
Fluorochrome or enzyme conjugate enzyme conjugation HRP
12) Washing after primary and secondary antibodies:
Buffer PBS pH 7,3
Number of washes 3 times 5 minutes
13) Detection method Diaminobenzidin tablets ( Kit by Sigma ), kept in freezer -20°C as recommended
14) How many times have you run this staining? 2 times
Do you obtain the same results every time? Partially, now the RAG-mouse slides seem to be clean, the Wildtype slides stay "suboptimal"
What steps have you altered to try and optimize the use of this antibody?
- changing the blocking solution concentration from 2% to 5%
- changing the time of peroxidase blocking from 15 to 30 minutes
- trying a goat-anti-mouse-fc-block from affini pure 1:1000 to exclude fc-binding
- testing a new ordered preadsorbed antibody

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Answer

Thank you for taking the time to complete our questionnaire and contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from this antibody.

The details you have kindly provided will enable us to investigate this case for you and this is also helpful in our records for monitoring of quality.

In the event that a product is not functioning in the applications cited on the product data sheet, we will be pleased to provide a credit note or free of charge replacement.

The antibody shows only background staining if there is endohenous Ig in the sample.

Based on the images and the accompanying information, Iwould like tothe following recommendations:

1) use thinner tissue sections; our standard recommendation for tissue of this type is 2-3um - you cite using 4-6um, which may be causing issues with background staining,

2) use greatly reduced fixation time; our recommendation for this type of tissue is less than 24hours, but half that time should be sufficient to obtain very good results,

3) use higher concentration blocking buffer; our recommendation is to use at least 10% serum.

I hope this information is helpful, thank you for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again.

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Question

Sehr geehrte Damen und Herren,
wir hinterlegen zu dem demnächst zu bestellenden Produkt automatisch Datasheet und SDS für den obengenannten Antikörper.
Diese sind leider nicht über Ihre homepage aufrufbar. Es erscheinen lediglich leere Seiten.
Bitte ergänzen Sie folgende .pdf-Dateien
https://www.abcam.com/Goat-polyclonal-Secondary-Antibody-to-Rat-IgG-H-L-HRP-pre-adsorbed-ab98425.pdf
https://www.abcam.com/ps/Products/98/ab98425/Msds/ab98425_CLP1_DE.pdf
Mit freundlichen Grüßen

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Answer

Vielen Dank für Ihre Email.

Die Links für das Datenblatt in PDF und das Material Safety Data Sheet funktionieren vom meinem Computer. Unsere IT Abteilung konnte auch keinen Fehler finden. Bitte überprüfen Sie die Einstelllungen an Ihrem Computer.

Ich habe beide Dokumente zu Ihrer Information dieser Email als Anhang beigefügt.

Bitte entschuldigen Sie die Unannehmlichkeiten und zögern Sie nicht mich nochmals zu kontaktieren falls Sie weitere Fragen haben.

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