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    progesterone-receptor-antibody-alpha-pr6-ab2765.pdf

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Epigenetics and Nuclear Signaling Transcription Domain Families Zinc Finger
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Anti-Progesterone Receptor 抗体 [Alpha PR6] (ab2765)

  • Datasheet
  • SDS
Reviews (5)Q&A (7)References (24)

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出荷および受注について

Abpromise

保証された製品品質、優れたカスタマー・サポート。 詳細はこちら。

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Progesterone Receptor antibody [Alpha PR6] (ab2765)
  • Immunocytochemistry/ Immunofluorescence - Anti-Progesterone Receptor antibody [Alpha PR6] - ChIP Grade (ab2765)
  • Western blot - Anti-Progesterone Receptor antibody [Alpha PR6] - ChIP Grade (ab2765)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Progesterone Receptor antibody [Alpha PR6] (ab2765)
  • Flow Cytometry - Anti-Progesterone Receptor antibody [Alpha PR6] - ChIP Grade (ab2765)

Key features and details

  • Mouse monoclonal [Alpha PR6] to Progesterone Receptor
  • Suitable for: IHC-P, WB, Flow Cyt, ICC/IF
  • Reacts with: Mouse, Rat, Sheep, Rabbit, Chicken, Guinea pig, Cow, Human
  • Isotype: IgG2a

こちらの製品もご検討ください

二次抗体
Product image
Goat Anti-Mouse IgG H&L (HRP) (ab205719)
標識
Product image
Alexa Fluor® 488 Conjugation Kit (Fast) - Lightning-Link® (ab236553)

関連製品

製品の概要

  • 製品名

    Anti-Progesterone Receptor antibody [Alpha PR6]
    Progesterone Receptor 一次抗体 製品一覧
  • 製品の詳細

    Mouse monoclonal [Alpha PR6] to Progesterone Receptor
  • 由来種

    Mouse
  • 特異性

    Detects the B form of the progesterone receptor (PR). This antibody does not cross-react with estrogen receptor or glucocorticoid receptor.
  • アプリケーション

    適用あり: IHC-P, WB, Flow Cyt, ICC/IFmore details
  • 種交差性

    交差種: Mouse, Rat, Sheep, Rabbit, Chicken, Guinea pig, Cow, Human
  • 免疫原

    Other Immunogen Type corresponding to Chicken Progesterone Receptor. Progesterone receptor purified from chick oviduct cytosol.

  • 特記事項

    Reproducibility is key to advancing scientific discovery and accelerating scientists’ next breakthrough.

    Abcam is leading the way with our range of recombinant antibodies, knockout-validated antibodies and knockout cell lines, all of which support improved reproducibility.

    We are also planning to innovate the way in which we present recommended applications and species on our product datasheets, so that only applications & species that have been tested in our own labs, our suppliers or by selected trusted collaborators are covered by our Abpromise™ guarantee.

    In preparation for this, we have started to update the applications & species that this product is Abpromise guaranteed for.

    We are also updating the applications & species that this product has been “predicted to work with,” however this information is not covered by our Abpromise guarantee.

    Applications & species from publications and Abreviews that have not been tested in our own labs or in those of our suppliers are not covered by the Abpromise guarantee.

    Please check that this product meets your needs before purchasing. If you have any questions, special requirements or concerns, please send us an inquiry and/or contact our Support team ahead of purchase. Recommended alternatives for this product can be found below, as well as customer reviews and Q&As.

製品の特性

  • 製品の状態

    Liquid
  • 保存方法

    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.
  • バッファー

    Preservative: 0.05% Sodium azide
    Constituent: PBS
  • Concentration information loading...
  • 精製度

    Protein G purified
  • ポリ/モノ

    モノクローナル
  • クローン名

    Alpha PR6
  • アイソタイプ

    IgG2a
  • 研究分野

    • Epigenetics and Nuclear Signaling
    • Transcription
    • Domain Families
    • Zinc Finger
    • Signal Transduction
    • Signaling Pathway
    • Nuclear Signaling
    • Nuclear Hormone Receptors
    • Progesterone
    • Neuroscience
    • Endocrine system
    • Gonadotrophic axis
    • Epigenetics and Nuclear Signaling
    • Nuclear Signaling Pathways
    • Nuclear Receptors
    • Progesterone
    • Cancer
    • Signal transduction
    • Nuclear signaling
    • Nuclear hormone receptors
    • Progesterone
    • Cancer
    • Tumor biomarkers
    • Receptors

関連製品

  • ChIP Related Products

    • ChIP Kit (ab500)
  • Compatible Secondaries

    • Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) (ab150113)
    • Goat Anti-Mouse IgG H&L (HRP) (ab205719)
    • Goat Anti-Mouse IgG H&L (DyLight® 488) preadsorbed (ab96879)
  • Conjugation kits

    • PE / R-Phycoerythrin Conjugation Kit - Lightning-Link® (ab102918)
    • APC Conjugation Kit - Lightning-Link® (ab201807)
  • Isotype control

    • Mouse IgG2a, kappa monoclonal [MG2a-53] - Isotype control (ab18415)
  • Recombinant Protein

    • Recombinant Human Progesterone Receptor protein (ab159105)

アプリケーション

Our Abpromise guarantee covers the use of ab2765 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 Use a concentration of 5 µg/ml.
WB Use a concentration of 1 µg/ml. Predicted molecular weight: 99 kDa.
Flow Cyt Use 0.5µg for 106 cells.

ab170191 - Mouse monoclonal IgG2a, is suitable for use as an isotype control with this antibody.

ICC/IF Use at an assay dependent concentration.

ターゲット情報

  • 機能

    The steroid hormones and their receptors are involved in the regulation of eukaryotic gene expression and affect cellular proliferation and differentiation in target tissues. Progesterone receptor isoform B (PRB) is involved activation of c-SRC/MAPK signaling on hormone stimulation.
    Isoform A: inactive in stimulating c-Src/MAPK signaling on hormone stimulation.
    Isoform 4: Increases mitochondrial membrane potential and cellular respiration upon stimulation by progesterone.
  • 配列類似性

    Belongs to the nuclear hormone receptor family. NR3 subfamily.
    Contains 1 nuclear receptor DNA-binding domain.
  • ドメイン

    Composed of three domains: a modulating N-terminal domain, a DNA-binding domain and a C-terminal ligand-binding domain.
  • 翻訳後修飾

    Phosphorylated on multiple serine sites. Several of these sites are hormone-dependent. Phosphorylation on Ser-294 occurs preferentially on isoform B, is highly hormone-dependent and modulates ubiquitination and sumoylation on Lys-388. Phosphorylation on Ser-102 and Ser-345 also requires induction by hormone. Basal phosphorylation on Ser-81, Ser-162, Ser-190 and Ser-400 is increased in response to progesterone and can be phosphorylated in vitro by the CDK2-A1 complex. Increased levels of phosphorylation on Ser-400 also in the presence of EGF, heregulin, IGF, PMA and FBS. Phosphorylation at this site by CDK2 is ligand-independent, and increases nuclear translocation and transcriptional activity. Phosphorylation at Ser-162 and Ser-294, but not at Ser-190, is impaired during the G(2)/M phase of the cell cycle. Phosphorylation on Ser-345 by ERK1/2 MAPK is required for interaction with SP1.
    Sumoylation is hormone-dependent and represses transcriptional activity. Sumoylation on all three sites is enhanced by PIAS3. Desumoylated by SENP1. Sumoylation on Lys-388, the main site of sumoylation, is repressed by ubiquitination on the same site, and modulated by phosphorylation at Ser-294.
    Ubiquitination is hormone-dependent and represses sumoylation on the same site. Promoted by MAPK-mediated phosphorylation on Ser-294.
    Palmitoylated by ZDHHC7 and ZDHHC21. Palmitoylation is required for plasma membrane targeting and for rapid intracellular signaling via ERK and AKT kinases and cAMP generation.
  • 細胞内局在

    Nucleus. Cytoplasm. Nucleoplasmic shuttling is both homone- and cell cycle-dependent. On hormone stimulation, retained in the cytoplasm in the G(1) and G(2)/M phases; Mitochondrion outer membrane and Nucleus. Cytoplasm. Mainly nuclear.
  • Target information above from: UniProt accession P06401 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • 参照データベース

    • Entrez Gene: 396198 Chicken
    • Entrez Gene: 100848466 Cow
    • Entrez Gene: 5241 Human
    • Entrez Gene: 18667 Mouse
    • Entrez Gene: 100009094 Rabbit
    • Entrez Gene: 25154 Rat
    • Omim: 607311 Human
    • SwissProt: P06401 Human
    • SwissProt: Q00175 Mouse
    • SwissProt: Q63449 Rat
    • Unigene: 32405 Human
    • Unigene: 742403 Human
    • Unigene: 12798 Mouse
    • Unigene: 437703 Mouse
    • Unigene: 10303 Rat
    see all
  • 別名

    • NR3C3 antibody
    • Nuclear receptor subfamily 3 group C member 3 antibody
    • PGR antibody
    • PR antibody
    • PRA antibody
    • PRB antibody
    • PRGR_HUMAN antibody
    • Progesterone receptor antibody
    • Progestin receptor form A antibody
    • Progestin receptor form B antibody
    see all

画像

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Progesterone Receptor antibody [Alpha PR6] (ab2765)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Progesterone Receptor antibody [Alpha PR6] (ab2765)

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) was performed on human uterus tissue. Antigen retrieval was performed using 10mM sodium citrate followed by microwave treatment for 8-15 minutes. Endogenous peroxidases were blocked in 3% H202-methanol for 15 minutes and tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Cells were incubated with ab2765 (1:20) overnight in a humidified chamber. Tissues were washed in PBST and detection was performed using a secondary antibody conjugated to HRP. DAB staining buffer was applied and tissues were counterstained with hematoxylin and prepped for mounting. Images were taken at 40X magnification.

  • Immunocytochemistry/ Immunofluorescence - Anti-Progesterone Receptor antibody [Alpha PR6] - ChIP Grade (ab2765)
    Immunocytochemistry/ Immunofluorescence - Anti-Progesterone Receptor antibody [Alpha PR6] - ChIP Grade (ab2765)

    Immunocytochemistry/ Immunofluorescence analysis of T47D cells untreated (left) or stimulated with 100nm promegestone for 1 hour (right), labeling Progesterone Receptor with ab2765 (green). The cells were fixed with formalin for 15 minutes, permeabilized with 0.1% Triton X-100 in TBS for 10 minutes, and blocked with 3% Blocker BSA for 15 minutes at room temperature. Cells were stained with Anti-Progesterone Receptor antibody [Alpha PR6] - ChIP Grade (ab2765) at a dilution of 1/100 for 1 hour at 37C, and then incubated with a Alexa Fluor 488 goat anti-mouse IgG secondary antibody at a dilution of 1/1000 for 30 minutes at room temperature (both panels, green). Nuclei (both panels, blue) were stained with Hoechst 33342 dye.

  • Western blot - Anti-Progesterone Receptor antibody [Alpha PR6] - ChIP Grade (ab2765)
    Western blot - Anti-Progesterone Receptor antibody [Alpha PR6] - ChIP Grade (ab2765)
    All lanes : Anti-Progesterone Receptor antibody [Alpha PR6] (ab2765) at 1 µg/ml

    Lane 1 : T47D cell lysate untreated (-)
    Lane 2 : T47D cell lysate stimulated (+) with 100 nm promegestone for 1 hour

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat anti-Mouse IgG-HRP at 1/2000 dilution

    Predicted band size: 99 kDa

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Progesterone Receptor antibody [Alpha PR6] (ab2765)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Progesterone Receptor antibody [Alpha PR6] (ab2765)This image is courtesy of Takako Akamatsu, Kansai Medical University.
    ab2765 staining Progesterone Receptor in Rat mammary tissue by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded tissue sections). The sections were fixed in formaldehyde and subjected to heat-mediated antigen retrieval by pressure cooker prior to blocking with 5% BSA for 5 minutes at room temperature. The primary antibody was diluted 1/100 in TBST and incubated with the sample for 16 hours at 4°C. A biotin-conjugated goat anti-mouse polyclonal was used as the secondary antibody.

    See Abreview

  • Flow Cytometry - Anti-Progesterone Receptor antibody [Alpha PR6] - ChIP Grade (ab2765)
    Flow Cytometry - Anti-Progesterone Receptor antibody [Alpha PR6] - ChIP Grade (ab2765)
    Overlay histogram showing T47D cells stained with ab2765 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab2765, 0.5µg/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 IgG2a [ICIGG2A] (ab91361, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.

プロトコール

  • Flow cytometry protocols
  • Immunohistochemistry protocols
  • Immunocytochemistry & immunofluorescence protocols
  • Western blot protocols

Click here to view the general protocols

データシートおよび資料

    • Datasheet
    • SDS
  • 参考文献 (24)

    ab2765 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。

    ab2765 は 24 報の論文で使用されています。

    • Tiemann TT  et al. Towards uterus tissue engineering: a comparative study of sheep uterus decellularisation. Mol Hum Reprod 26:167-178 (2020). PubMed: 31980817
    • Kim YY  et al. Effects of Natural Progesterone and Synthetic Progestin on Germ Layer Gene Expression in a Human Embryoid Body Model. Int J Mol Sci 21:N/A (2020). PubMed: 31991577
    • Xiao L  et al. Dihydrotestosterone synthesis in the sheep corpus luteum and its potential mechanism in luteal regression. J Cell Physiol N/A:N/A (2019). PubMed: 30671954
    • Park SY  et al. Distribution of and steroid hormone effects on calbindin-D9k in the immature rat brain. Brain Res Bull 152:225-235 (2019). PubMed: 31357009
    • Jin X  et al. ERa is required for suppressing OCT4-induced proliferation of breast cancer cells via DNMT1/ISL1/ERK axis. Cell Prolif 52:e12612 (2019). PubMed: 31012189
    View all Publications for this product

    レビューと Q&A

    Show All レビュー Q&A
    レビューを送る 質問を送る

    1-10 of 12 Abreviews or Q&A

    Immunocytochemistry/ Immunofluorescence abreview for Anti-Progesterone Receptor antibody [Alpha PR6] - ChIP Grade

    Good
    Abreviews
    Abreviews
    abreview image
    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Sheep Cell (fetal sheep cells)
    Permeabilization
    No
    Specification
    fetal sheep cells
    Blocking step
    Serum as blocking agent for 30 minute(s) · Concentration: 10% · Temperature: RT°C
    Fixative
    Formaldehyde
    Read More

    Abcam user community

    Verified customer

    投稿 Jan 29 2020

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) abreview for Anti-Progesterone Receptor antibody [Alpha PR6] - ChIP Grade

    Good
    Abreviews
    Abreviews
    abreview image
    Application
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
    Sample
    Sheep Tissue sections (sheep uterus)
    Antigen retrieval step
    Heat mediated - Buffer/Enzyme Used: 10mM Citric acid,pH 6.0, 0,05% Tween 20
    Permeabilization
    Yes - Triton-X100 0.2%
    Specification
    sheep uterus
    Blocking step
    Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: RT°C
    Fixative
    Formaldehyde
    Read More

    Abcam user community

    Verified customer

    投稿 Jan 29 2020

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) abreview for Anti-Progesterone Receptor antibody [Alpha PR6] - ChIP Grade

    Poor
    Abreviews
    Abreviews
    abreview image
    Application
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
    Sample
    Dog Tissue sections (Uterus)
    Antigen retrieval step
    Heat mediated
    Permeabilization
    No
    Specification
    Uterus
    Blocking step
    Serum as blocking agent for 30 minute(s) · Concentration: 10% · Temperature: 23°C
    Fixative
    Formaldehyde
    Read More

    Abcam user community

    Verified customer

    投稿 Jun 21 2019

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) abreview for Anti-Progesterone Receptor antibody [Alpha PR6] - ChIP Grade

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
    Sample
    Mouse Tissue sections (placenta)
    Antigen retrieval step
    Heat mediated - Buffer/Enzyme Used: 10 mM citrate, pH6.0
    Permeabilization
    No
    Specification
    placenta
    Blocking step
    Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 25°C
    Fixative
    Formaldehyde
    Read More

    Abcam user community

    Verified customer

    投稿 Sep 01 2015

    Question

    Vielen Dank für Ihre schnelle Antwort auf meine Anfrage! Leider ist die vollständige Aminosäuresequenz des Fusionsproteins in der Literatur nicht zu finden.

    Der Beschreibung nach handelt es sich um Cre-Rekombinase (AS-Sequenz: http://www.uniprot.org/uniprot/P06956 ?) kombiniert mit einem „nuclear localisation signal“ (Pro-Lys-Lys-Lys-Arg-Lys-Val).

    Hinzu kommt eine mutierte Form (hPR891) der „hormone-binding domain“ (HBD; aa 641–891) des humanen Progesteronrezeptors, bei welcher die C-terminalen 42 Aminosäuren fehlen. Diese hätte also vermutlich folgende AS-Sequenz:

    FNKVRVVRA LDAVALPQPV GVPNESQALS QRFTFSPGQD IQLIPPLINL LMSIEPDVIY AGHDNTKPDT SSSLLTSLNQ LGERQLLSVV KWSKSLPGFR NLHIDDQITL IQYSWMSLMV FGLGWRSYKH VSGQMLYFAP DLILNEQRMK ESSFYSLCLT MWQIPQEFVK LQVSQEEFLC MKVLLLLNTI PLEGLRSQTQ FEEMRSSYI

    Unter diesen Voraussetzungen, wäre es Ihrer Einschätzung nach sicherer einen der vorhandenen Progesteronrezeptor-Antikörper zu verwenden, oder einen Cre-Antikörper? Der Antikörper soll zur immunhistochemischen Färbung von cryofixiertem Gewebe transgener Mäuse verwendet werden.

    Haben Sie herzlichen Dank für Ihre Unterstützung!


    Mit freundlichen Grüßen

    Read More

    Abcam community

    Verified customer

    Asked on Dec 04 2012

    Answer

    Vielen Dank für diese zusätzliche Information.

    Leider haben wir keinen Antikörper den ich für die Detektion Ihres rekombinanten Proteins garantieren kann. Ich habe drei Antikörper gefunden die vielleicht für Sie in Frage kommen und die Sie sich genauer anschauen sollten.

    1. https://www.abcam.com/cre-recombinase-antibody-723-ab24607.html

    Immunogensequenz ist das gesamte native Cre Rekombinase Protein. Die Sequenz sollte daher mit der Sequenz in dem rekombinanten Protein übereinstimmen.

    2. https://www.abcam.com/cre-recombinase-antibody-ab137240.html

    Bei dem Immunogen handelt es sich um ein rekombinantes full length protein des Bacteriophage P1 Cre recombinase.

    3. https://www.abcam.com/progesterone-receptor-antibody-alpha-pr6-ab2765.html

    Die Immunogensequenz dieses Antikörpers ist leider nicht näher bestimmt. Der Antikörper wurde gegen das gesamte Hühnchen Progesteron gerichtet hergestellt. Die Hühnchensequenz verglichen mit der zur Verfügung gestellten Progesteronsequenz des rekombinanten Proteins weißt eine Uebereinstimmung von 78% auf. Die Detektion eines Proteins werten wir als wahrscheinlich, wenn eine Übereinstimmung von etwa 85% besteht.



    Der ab24607 ist unter diesen drei Möglichkeiten der mit den meisten Referenzen und eingereichten Abreviews und daher am besten charakterisiert. Ab24607 wurde schon für IHC-Fr in Maus verwendet und zwei Abreviews speziell hierfür wurden verfasst.

    Ich hoffe ich konnte Ihnen etwas weiter helfen. Es tut mir Leid dass ich Ihnen in diesem Fall kein perfektes Produkt anbieten kann.

    Sollten Sie sich dafür entscheiden einen dieser Antikörper zu kaufen, möchte ich Sie noch auf unsere RabMab Aktion aufmerksam machen. Bitte lesen Sie mehr über diese Aktionhttps://www.abcam.com/index.html?pageconfig=resource&rid=15447.

    Bitte zögern Sie nicht sich wieder bei mir zu melden, sollten Sie weitere Fragen oder Probleme mit unseren Produkten haben.

    Read More

    Abcam Scientific Support

    Answered on Dec 04 2012

    Question

    What is the volume in ul for ab2765

    Read More

    Abcam community

    Verified customer

    Asked on Jun 12 2012

    Answer

    Thank you for contacting us.

    It will be 100ul.

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

    Use our products? Submit an Abreview. Earn rewards!
    https://www.abcam.com/abreviews

    Read More

    Abcam Scientific Support

    Answered on Jun 12 2012

    Question

    storage buffer composition? Ab purified?

    Read More

    Abcam community

    Verified customer

    Asked on Dec 09 2011

    Answer

    Yes, the antibody is purified. The solution before lyophilization is 1X PBS with 0.05% sodium azide. I hope this is helpful. Please contact us again if you have any further questions.

    Read More

    Abcam Scientific Support

    Answered on Dec 09 2011

    Question

    storage buffer composition? Ab purified?

    Read More

    Abcam community

    Verified customer

    Asked on Dec 07 2011

    Answer

    Thank you for contacting us. I can confirm that the storage buffer for this antibody is PBS containing 0.05% sodium azide, and that it is purified from mouse ascites. Should I receive any further information from the lab, then I will let you know. I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

    Read More

    Abcam Scientific Support

    Answered on Dec 07 2011

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) abreview for Anti-Progesterone Receptor antibody [Alpha PR6]

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
    Sample
    Rat Tissue sections (mammary carcinoma)
    Specification
    mammary carcinoma
    Fixative
    Formaldehyde
    Antigen retrieval step
    Heat mediated - Buffer/Enzyme Used: pressure cooker
    Permeabilization
    No
    Blocking step
    BSA as blocking agent for 5 minute(s) · Concentration: 5% · Temperature: RT°C
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    孝子 赤松

    Verified customer

    投稿 Sep 27 2011

    Question

    BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER 217040 DESCRIPTION OF THE PROBLEM Non-specific band.Th strongest band located at 50kDa and second strong band is at 23 KDa. No band show at 99KDa. SAMPLE protein from kidney cortex PRIMARY ANTIBODY overnight at 4C DETECTION METHOD LumiGLO POSITIVE AND NEGATIVE CONTROLS USED No ANTIBODY STORAGE CONDITIONS -20C SAMPLE PREPARATION Homogenization Buffer( 40mM Tris HCl,40mM Tris Base) with 1:100 Protease inhibitor Cocktail Set III(Calbiochem#539134). at 95C for 5min and then on ice AMOUNT OF PROTEIN LOADED 23ug ELECTROPHORESIS/GEL CONDITIONS Reducing ,15% gel TRANSFER AND BLOCKING CONDITIONS taransfer at 0.5 A for 1 hour. Block 1hour with 5% milk. SECONDARY ANTIBODY 1:10000 for 1 hour HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 1 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? No

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    Abcam community

    Verified customer

    Asked on Apr 27 2007

    Answer

    Thank you for your enquiry. I am sorry to hear that you have been experiencing problems with ab2675 in western blot. Often it is possible to make suggestions that help resolve problems. We will happily offer technical support and in the event that a product is not functioning in the applications cited on the product data sheet (and the problem has been reported within 90 days of purchase), and if it appears that the antibody is at fault, a credit note/refund will be offered. I have looked through your protocols and they seem perfectly fine to me. However, I have a few questions that I hope you can answer so that I can understand the problem better. 1) I might not know much about this field but is the expression of progesterone receptor found to be high in the kidney cortex? I would assume that the bands you observed are non-specific. Please try using a positive control like oviduct of chicks with your samples. You can also refer to the references found on the datasheet of this product for more information. 2) Can you please confirm the species from which you are extracting the kidney cortex protein from? This product has only been in tested in Human, Rabbit, Rat, Mouse, Cow, Guinea Pig and Chicken. 3) What was the amount of primary antibody you used to incubate the blot? We would normally suggest 1:300-1:3000 but it is up to the end user to optimize the results. Also, what was the buffer you used to incubate the primary and secondary antibodies? 4) You can also try using a lower percentage gel, like 8-10%, as the protein size of this antibody is expected to be around 99kDa. 5) You mentioned that you used reduced condition. Can you confirm that you also included SDS and beta-mercaptoethanol in the buffer and at what concentration? If the above suggestions did you help you resolve your problem, please get back to me with an image of your immunoblot and purchase details (lot number, shipping address, etc). Should you require any further information or assistance, please do not hesitate to contact me.

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    Abcam Scientific Support

    Answered on Apr 27 2007

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