Overview

  • Product name
    Anti-GnRHR antibody [A9E4]
    See all GnRHR primary antibodies
  • Description
    Mouse monoclonal [A9E4] to GnRHR
  • Host species
    Mouse
  • Specificity
    It is unknown if ab24095 reacts with the receptor form 1 or 2.
  • Tested applications
    Suitable for: IHC-Fr, Flow Cyt, ELISA, WBmore details
  • Species reactivity
    Reacts with: Human
    Predicted to work with: Rabbit, Pig
  • Immunogen

    Synthetic peptide:

    MANSASPEQNQHCSAINNSIPLMQGNLPY

    conjugated to BSA, corresponding to N terminal amino acids 1-29 of Human GnRHR

  • Positive control
    • IHC-Fr: human anterior pituitary sections WB/FACS: GT7-1 cells (hypothalamus cell line)

Properties

  • Form
    Liquid
  • Storage instructions
    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term.
  • Storage buffer
    Preservative: 0.05% Sodium azide
    Constituent: PBS
  • Concentration information loading...
  • Purity
    Protein G purified
  • Clonality
    Monoclonal
  • Clone number
    A9E4
  • Myeloma
    Sp2/0
  • Isotype
    IgG1
  • Light chain type
    kappa
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab24095 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-Fr 1/20 - 1/40.
Flow Cyt Use 1µg for 106 cells.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

ELISA 1/100.
WB 1/20 - 1/40. Predicted molecular weight: 38 kDa.

Target

  • Information by UniProt
  • Database links
  • Alternative names
    • GnRH receptor antibody
    • GnRH-R antibody
    • GNRHR antibody
    • GNRHR_HUMAN antibody
    • GNRHR1 antibody
    • Gonadotropin releasing hormone receptor antibody
    • gonadotropin-releasing hormone (type 1) receptor 1 antibody
    • Gonadotropin-releasing hormone receptor antibody
    • GRHR antibody
    • HH7 antibody
    • leutinizing-releasing hormone receptor antibody
    • lh-rh antibody
    • LHRHR antibody
    • LRHR antibody
    • luliberin receptor antibody
    • luteinizing hormone releasing hormone receptor antibody
    • type I GnRH receptor antibody
    see all

Images

  • Overlay histogram showing MCF7 cells stained with ab24095 (red line). The cells were fixed with 80% methanol (5 min) and then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab24095, 1µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells ) used under the same conditions. Acquisition of >5,000 events was performed.
    Please note that Abcam do not have any data for use of this antibody on non-fixed cells. We welcome any customer feedback.

References

This product has been referenced in:
  • Xu B  et al. Increased Uterine NK cell numbers and perforin expression during the implantation phase in IVF Cycles with GnRH Antagonist Protocol. Sci Rep 7:39912 (2017). IHC . Read more (PubMed: 28045093) »
  • Seitz S  et al. Triple negative breast cancers express receptors for LHRH and are potential therapeutic targets for cytotoxic LHRH-analogs, AEZS 108 and AEZS 125. BMC Cancer 14:847 (2014). IHC ; Human . Read more (PubMed: 25410881) »
See all 2 Publications for this product

Customer reviews and Q&As

1-4 of 4 Abreviews or Q&A

Answer

Usually if you are trying to replicate our results, we first suggest trying the specific protocol listed on our datasheet.

For this antibody, we tested it in our lab under the conditions stated on the datasheet and this gave good results in our hands. Aquisition of data was using a Cytomics FC500 MPL Flow Cytometer (Beckman Coulter). Filter used: 525nm BandPass (PMT1). Please note that PFA MCF7 cells gave minimal positive signal above the negative isotype control using the stated concentration/dilution. PFA 61% signal. Previous dilutions: 1/20-1/40.

We have no reason to believe that a standard FACS protocol won't give positive results but like all experiments, it is a question of trial and improvement hence why we usually suggest our customers use the same conditions as stated on the datasheet and then optimise as necessary.

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Question

1) Abcam product code  ab24095                     2) Abcam order reference number or product batch number      lot: GR29659-1   3) Description of the problem     antibody binds unspecific, no specific band at 38 or 68 kDa   4) Sample preparation: Type of sample (whole cell lysates, fraction, recombinant protein…): whole cell lysates Lysis buffer RIPA    (sonification) Protease inhibitors: Roche Protease Inhibitor Cocktail, PMSF Phosphatase inhibitors Reducing agent DTT      Boiling for ≥5 min? 5min  Protein loaded ug/lane or cells/lane 30µg and 100µg tested Positive control DU-145 cells overexpressing GnRHR Negative control DU-145 empty vector control and HEK293T   5) Percentage of gel 10% Type of membrane PVDF Protein transfer verified   Ponceau           Blocking agent and concentration 5% BSA in PBS-Tween Blocking time                1h                    Blocking temperature RT   6) Primary antibody (If more than one was used, describe in “additional notes”) : Concentration or dilution 1:40  Diluent buffer  TBS-T with 3% BSA Incubation time o/n Incubation temperature: 4°C   7)  Secondary antibody: Species: sheep Reacts against: mouse IgG Concentration or dilution 1:10000 Diluent buffer  5% Milk in PBS-Tween Incubation time 1h Incubation temperature: RT Fluorochrome or enzyme conjugate: peroxidase   8) Washing after primary and secondary antibodies: Buffer PBS-Tween Number of washes 3-4 (1h in total)           9)Detection method chemoluminescence 10) How many times have you run this staining? 2 times Do you obtain the same results every time?  no What steps have you altered to try and optimize the use of this antibody? different protein concentrations

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Answer

Vielen Dank, dass Sie sich die Zeit genommen haben, unseren Fragebogen auszufüllen und ein Bild mitzuschicken. Es tut mir leid, dass Sie Probleme mit unserem Antikörper hatten. Um Ihr Problem zu lösen, möchte ich die folgenden Vorschläge zu Veränderung Ihres Protokolls machen: 1.) Ich schlage vor, eine "no primary"- Kontrolle durchzuführen, um auszuschließen, dass die falschen Banden vom sekundären Antikörper hervorgerufen werden. 2.) In manchen Fällen kann die Verwendung von Milch zum Blockieren den Hintergrund verringern. Außerdem habe ich noch ein paar Fragen und Anmerkungen: 1.) Es gibt zwei Formen dieses Rezeptors. Wurde überprüft, ob die überexprimierte Form die Immunogen -Sequenz enthält? 2.) Ich kann auf dem Blot eine Doppelbande in der Höhe von ~38kDa erkennen, die nur in der Prostata Zelllinie vorhanden ist. Dafür habe ich folgende mögliche Erklärungen: A: Diese Ziellinie enthält endogenen GnRHR. B: Die Proben wurden vertauscht und die stärkere Bande ist tatsächlich überexprimerter GnRHR. C: HEK293 Zellen sollten keinen endogenen GnRHR exprimieren und deshalb ist keine Bande zu sehen. GFP ist ein sehr großer Tag und verhindert oft, dass ein Antikörper das Protein noch erkennen kann, da das Protein nicht mehr richtig gefaltet wird. Wir können leider nicht garantieren, dass unsere Antikörper getaggte Proteine erkennen. Bitte lassen Sie mich wissen, ob ich Ihnen helfen konnte und zögern Sie nicht, sich wieder bei uns zu melden, falls Sie weitere Fragen haben und meine Vorschläge nicht zu einer Klärung des Problems führen.

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Answer

Thank you for your enquiry. The molecular weight that we have stated on our datasheet is the predicted molecular weight, base on Swiss Prot database. The actual molecular weight could be higher or lower and depends on factors like different modifications of glycosylation. I have managed to find a literature relating to a clone and have listed it below for your reference. Unfortunately, due to my limited access to journal, I was unable to check if the literature have any answers to your question but I hope it will provide some useful information to you. Karande, A.A., et al., Establishment of immunological probes to study human gonadotropin-releasing hormone receptors. Molec.Cell Endocrinol.114, 51-56, 1995 Should you require any further assistance, please feel free to contact me.

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Answer

Thank you for your enquiry. The immunogen sequence used to generate the antibody ab24095 is MANSASPEQNQHCSAINNSIPLMQGNLPY and a BLAST search showed 96% homology with both the isoforms 1 and 2, therefore we do not know if ab24095 reacts with receptor 1 or receptor 2 or both. Both ab24095 and ab24098 work very well on FACS and have been tested on GT7-1 cells (hypothalamus cell line) but not on T lymphocytes. Please let me know if you require further information,

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