Recombinant
RabMAb

Anti-JAK2 (phospho Y1007 + Y1008) antibody [E132] (ab32101)

Overview

  • Product name
    Anti-JAK2 (phospho Y1007 + Y1008) antibody [E132]
    See all JAK2 primary antibodies
  • Description
    Rabbit monoclonal [E132] to JAK2 (phospho Y1007 + Y1008)
  • Host species
    Rabbit
  • Specificity
    The antibody is phospho-specific and only detects phosphorylated JAK2 on Tyrosine 1007 and 1008 (pY1007+Y1008). According to our ELISA results, this antibody preferentially recognizes phospho Y1007. Stimulation may be required to allow detection of the phosphorylated protein. Please see images below for recommended treatment conditions and positive controls.
  • Tested applications
    Suitable for: ELISA, WB, IHC-Fr, ICC/IF, Flow Cyt, IHC-P, IP, Dot blotmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide (the amino acid sequence is considered to be commercially sensitive) within Human JAK2 (phospho Y1007 + Y1008). The exact sequence is proprietary.

  • Positive control
    • WB: Jurkat or K562 cells (treated with Pervanadate). IHC-P: Human lung adenocarcinoma tissue. ICC/IF: Jurkat cells (treated with Pervanadate). Flow Cyt: Jurkat starved of serum for 16 hours then treated with 1mM Pervanadate for 30 minutes.
  • General notes

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab32101 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
ELISA Use at an assay dependent concentration.
WB 1/1000 - 1/10000. Detects a band of approximately 120 kDa (predicted molecular weight: 130 kDa).

The samples may require stimulation (E.g., Jurkat cells treated with pervanadate for 5 min)

IHC-Fr Use at an assay dependent concentration. PubMed: 21420414
ICC/IF 1/1000.
Flow Cyt 1/20.
IHC-P Use at an assay dependent concentration.
IP 1/50.
Dot blot 1/1000.

Target

  • Function
    Non-receptor tyrosine kinase involved in various processes such as cell cycle progression, apoptosis, mitotic recombination, genetic instability and histone modifications. In the cytoplasm, plays a pivotal role in signal transduction via its association with cytokine receptors, which constitutes an initiating step in signaling for many members of the cytokine receptor superfamily including the receptors for growth hormone (GHR), prolactin (PRLR), leptin (LEPR), erythropoietin (EPOR), granulocyte-macrophage colony-stimulating factor (CSF2), thrombopoietin (THPO) and multiple interleukins. Following stimulation with erythropoietin (EPO) during erythropoiesis, it is autophosphorylated and activated, leading to its association with erythropoietin receptor (EPOR) and tyrosine phosphorylation of residues in the EPOR cytoplasmic domain. Also involved in promoting the localization of EPOR to the plasma membrane. Also acts downstream of some G-protein coupled receptors. Plays a role in the control of body weight (By similarity). Mediates angiotensin-2-induced ARHGEF1 phosphorylation. In the nucleus, plays a key role in chromatin by specifically mediating phosphorylation of 'Tyr-41' of histone H3 (H3Y41ph), a specific tag that promotes exclusion of CBX5 (HP1 alpha) from chromatin.
  • Tissue specificity
    Expressed in blood, bone marrow and lymph node.
  • Involvement in disease
    Note=Chromosomal aberrations involving JAK2 are found in both chronic and acute forms of eosinophilic, lymphoblastic and myeloid leukemia. Translocation t(8;9)(p22;p24) with PCM1 links the protein kinase domain of JAK2 to the major portion of PCM1. Translocation t(9;12)(p24;p13) with ETV6.
    Defects in JAK2 are a cause of susceptibility to Budd-Chiari syndrome (BCS) [MIM:600880]. It is a syndrome caused by obstruction of hepatic venous outflow involving either the hepatic veins or the terminal segment of the inferior vena cava. Obstructions are generally caused by thrombosis and lead to hepatic congestion and ischemic necrosis. Clinical manifestations observed in the majority of patients include hepatomegaly, right upper quadrant pain and abdominal ascites. Budd-Chiari syndrome is associated with a combination of disease states including primary myeloproliferative syndromes and thrombophilia due to factor V Leiden, protein C deficiency and antithrombin III deficiency. Budd-Chiari syndrome is a rare but typical complication in patients with polycythemia vera.
    Defects in JAK2 are a cause of polycythemia vera (PV) [MIM:263300]. A myeloproliferative disorder characterized by abnormal proliferation of all hematopoietic bone marrow elements, erythroid hyperplasia, an absolute increase in total blood volume, but also by myeloid leukocytosis, thrombocytosis and splenomegaly.
    Defects in JAK2 gene may be a cause of essential thrombocythemia (ET) [MIM:187950]. ET is characterized by elevated platelet levels due to sustained proliferation of megakaryocytes, and frequently lead to thrombotic and haemorrhagic complications.
    Defects in JAK2 are a cause of myelofibrosis (MYELOF) [MIM:254450]. Myelofibrosis is a disorder characterized by replacement of the bone marrow by fibrous tissue, occurring in association with a myeloproliferative disorder. Clinical manifestations may include anemia, pallor, splenomegaly, hypermetabolic state, petechiae, ecchymosis, bleeding, lymphadenopathy, hepatomegaly, portal hypertension.
    Defects in JAK2 are a cause of acute myelogenous leukemia (AML) [MIM:601626]. AML is a malignant disease in which hematopoietic precursors are arrested in an early stage of development.
  • Sequence similarities
    Belongs to the protein kinase superfamily. Tyr protein kinase family. JAK subfamily.
    Contains 1 FERM domain.
    Contains 1 protein kinase domain.
    Contains 1 SH2 domain.
  • Domain
    Possesses 2 protein kinase domains. The second one probably contains the catalytic domain, while the presence of slight differences suggest a different role for protein kinase 1.
  • Post-translational
    modifications
    Autophosphorylated, leading to regulate its activity. Leptin promotes phosphorylation on tyrosine residues, including phosphorylation on Tyr-813. Autophosphorylation on Tyr-119 in response to EPO down-regulates its kinase activity. Autophosphorylation on Tyr-868, Tyr-966 and Tyr-972 in response to growth hormone (GH) are required for maximal kinase activity.
  • Cellular localization
    Endomembrane system. Nucleus.
  • Information by UniProt
  • Database links
  • Alternative names
    • JAK 2 antibody
    • JAK-2 antibody
    • JAK2 antibody
    • JAK2_HUMAN antibody
    • Janus Activating Kinase 2 antibody
    • Janus kinase 2 (a protein tyrosine kinase) antibody
    • Janus kinase 2 antibody
    • JTK 10 antibody
    • JTK10 antibody
    • kinase Jak2 antibody
    • OTTHUMP00000043260 antibody
    • THCYT3 antibody
    • Tyrosine protein kinase JAK2 antibody
    • Tyrosine-protein kinase JAK2 antibody
    see all

Images

  • Immunocytochemistry/Immunofluorescence analysis of Jurkat +/- pervanadate (1mM, 30min) and Jurkat + pervanadate (1mM, 30min) + LP cells labelling JAK2 (phospho Y1007 + Y1008) with ab32101 at a dilution of 1/1000. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% tritonX-100. ab150077 (goat anti-rabbit IgG Alexa Fluor® 488) (1/1000) was used as the secondary antibody. The cells were co-stained with ab195889 (Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594)) at a 1/200 dilution. Nuclei counterstained with DAPI (blue).

  • All lanes : Anti-JAK2 (phospho Y1007 + Y1008) antibody [E132] (ab32101) at 1/5000 dilution

    Lane 1 : Untreated Jurkat cells whole cell lysates
    Lane 2 : Jurkat cells were treated with 50mM Pervanadate for 5 minutes whole cell lysates
    Lane 3 : Jurkat cells were treated with 50mM Pervanadate for 5 minutes whole cell lysates. Then the membrane was incubated with Alkaline phosphatase.

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

    Predicted band size: 130 kDa
    Observed band size: 120 kDa
    why is the actual band size different from the predicted?


    Exposure time: 5 seconds


    Blocking and diluting buffer 5% NFDM/TBST

  • ab32101 showing positive staining in Lung adenocarcinoma tissue.

  • Dot blot analysis of JAK2 (phospho Y1007 & Y1008) phospho peptide (Lane 1), JAK2 (phospho Y1007) phospho peptide (Lane 2), JAK2 (phospho Y1008) phospho peptide (Lane 3) and JAK2 non-phospho peptide (Lane 4) labelling JAK2 (phospho Y1007 & Y1008) with ab32101 at a dilution of 1/1000. A Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) was used as the secondary antibody at a dilution of 1/20,000. Blocking buffer: 5% NFDM/TBST. Dilution buffer: 5% NFDM /TBST.

  • All lanes : Anti-JAK2 (phospho Y1007 + Y1008) antibody [E132] (ab32101) at 1/1000 dilution

    Lane 1 : Jurkat (Human T cell leukemia T lymphocyte) whole cell lysates
    Lane 2 : Jurkat (Human T cell leukemia T lymphocyte) treated with 50mM Pervanadate for 5 minutes whole cell lysates

    Lysates/proteins at 15 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

    Predicted band size: 130 kDa
    Observed band size: 120 kDa why is the actual band size different from the predicted?
    Additional bands at: 60 kDa. We are unsure as to the identity of these extra bands.



    Blocking and diluting buffer: 5% NFDM/TBST

    Exposure time: 
    Left image: 1 second
    Right image: 5 minutes

  • Flow Cytometry analysis of Jurkat (human acute T cell leukemia) cells starved of serum for 16 hours then treated with 1 mM Pervanadate for 30 minutes labeling JAK2 (phospho Y1007 + Y1008) with ab32101 at 1/20 dilution (10 ug/mL) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor®488) at 1/2000 dilution was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) was used as the unlabeled control. 

  • Direct ELISA antigen dose-response curve using ab32101 at 0~1000 ng/mL. Antigen concentration of 100 ng/mL. An Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG(H+L) (1/2500) was used as the secondary antibody.

    This antibody preferentially recognizes phospho Y1007. When the concentration of peptides is higher than 100 ng/mL, it also recognizes phospho Y1008.

  • Direct ELISA antigen dose-response curve using ab32101 at 0~1000 ng/mL. Antigen concentration of 10 ng/mL. An Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG(H+L) (1/2500) was used as the secondary antibody.

    This antibody preferentially recognizes phospho Y1007. When the concentration of peptides is lower than 10 ng/mL, it cannot recognize phospho Y1008.

     

  • Analysis of immunogen phosphopeptide (A) and non phosphopeptide (B). Antibody used at 1/1000 dilution.
  • All lanes : Anti-JAK2 (phospho Y1007 + Y1008) antibody [E132] (ab32101) at 1/2000 dilution

    Lane 1 : Rat uterine cell line - whole cell lysate. Treated with 1µg/mL Prolactin for 0 hours.
    Lane 2 : Rat uterine cell line - whole cell lysate. Treated with 1µg/mL Prolactin for 15 minutes.
    Lane 3 : Rat uterine cell line - whole cell lysate. Treated with 1µg/mL Prolactin for 30 minutes.
    Lane 4 : Rat uterine cell line - whole cell lysate. Treated with 1µg/mL Prolactin for 1 hour.
    Lane 5 : Rat uterine cell line - whole cell lysate. Treated with 1µg/mL Prolactin for 2 hours.
    Lane 6 : Rat uterine cell line - whole cell lysate. Treated with 1µg/mL Prolactin for 4 hours.
    Lane 7 : Rat uterine cell line - whole cell lysate. Treated with 1µg/mL Prolactin for 6 hours.
    Lane 8 : Rat uterine cell line - whole cell lysate. Treated with 1µg/mL Prolactin for 24 hours.

    Lysates/proteins at 30 µg per lane.

    Secondary
    All lanes : An HRP-conjugated donkey anti-rabbit polyclonal. at 1/10000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 130 kDa
    Observed band size: 110 kDa why is the actual band size different from the predicted?
    Additional bands at: 55 kDa (possible non-specific binding)

    See Abreview

  • ab32101 showing positive staining in Ovarian carcinoma tissue.

  • ab32101 showing positive staining in Cervical carcinoma tissue.

  • ab32101 showing positive staining in Endometrial carcinoma tissue.

  • ab32101 showing positive staining in Urinary bladder transitional carcinoma tissue.

References

This product has been referenced in:
See all 40 Publications for this product

Customer reviews and Q&As

1-8 of 8 Abreviews or Q&A

Answer

Thank you for bringing this to our attention. I am sorry to read that two of the antibodies you purchased are not staining blots of your mouse liver samples.

I am assuming the transfer from gel to blot was effective, if your protocol worked with other blots of these samples.

I recommend stripping a blot and re-blocking with 1% BSA without detergent for one hour, and also diluting the antibodies in this 1% BSA solution, instead of non-fat dry milk. I also recommend trying the antibodies at higher concentrations, for instance 1/250 for anti-JAK2 ab39636, and 1/2000 for the phospho-Jak2 antibody, ab32101.

Please let me know the results. If this modification fails, I will be happy to provide replacements, or a credit or refund. The phosphorylation in the liver may be difficult to detect if it is not induced. If possible, I recommend including a positive control such as the treated mouse liver sample shown on the datasheet of ab68268.

Click here (or use the following: https://www.abcam.com/index.html?datasheet=68268).

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Question
Answer

Thank you for contacting us.

The concentration of lot GR57259-1 is XXXXXXX mg/ml.

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!
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Answer

Thank you for your enquiry.

I can confirm that this antibody has been tested on the following and these would be suitable positive controls:

Rat uterine cell line Treated with 1µg/mL Prolactin for 1 hour.

Jurkat cell lysate treated with GM-CSF (Granulocyte-macrophage colony-stimulating factor) or Pervanadate

I am sorry we do not provide lysates of these cell lines that have beenpre treated. Unfortunately, we did not test any cell lines that do not require pretreatment. I was not able to find positive controls for phosphorylated Jak2 in the literature, and from what I find stimulation is required for expression of this phosphorylated state of the protein in cell culture.

I can suggest to do a further literature search for more information.

I am sorry we have not suitable positive controls to provide from our catalog on this occasion, however I hope this information is useful to you. If you have any further questions, please do not hesitate to contact me.

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Question
Answer

Vielen Dank für Ihren Anruf und für Ihr Interesse an unseren Produkten. Gerne versichere ich Ihnen, dass alle unsere Produkte unsere Abcam Garantier unterliegen: Falls sich herausstellt, dass der Antikörper nicht so funktioniert, wie auf dem Datenblatt beschrieben und Sie sich innerhalb von sechs Monaten mit Ihrem Problem an uns wenden, werden wir Ihnen gerne Vorschläge zur Optimierung Ihres Protokolls oder gegebenenfalls einen Ersatz oder eine Gutschrift schicken. Wie am Telefon besprochen haben wir noch keine Abbildung von dem Antikörper ab32101 in der Immunhistochemie an Gefrierschnitten (IHC-Fr). Falls Sie uns eine Abbildung Ihrer Ergebnisse mit ab32101 zukommen lassen, kann ich Ihnen zurzeit ein spezielles Angebot über einen 100%igen Abreview-Rabatt anbieten. Bei diesem Angebot bekommen Sie einen Rabatt für eine zukünftige Bestellung, wenn Sie uns ein Abreview mit dem Testresultat und einer Abbildung zusenden. Der 100%ige Rabatt würde gegen eine erneute Bestellung eines primären Antikörpers von uns verrechnet werden. Um von diesem Angebot profitieren zu können, folgen Sie bitte diesen Schritten: 1.) Bestätigen Sie mir bitte, dass Sie ab32101 kaufen möchten und bereit sind uns eine Abbildung Ihrer Ergebnisse zukommen zu lassen. 2.) Bitte bestellen Sie erst nach Erhalt des Rabattcodes. 3.) Bestellen Sie den Antikörper wie üblich per Telefon, Email oder Fax 4.) Testen Sie den Antikörper in der IHC-Fr. 5.) Senden Sie uns die Abbildung Ihres Ergebnisses mittels eines Abreviews zu und notieren Sie den Discount Code in dem Feld "additional notes" Unter der folgenden URL können Sie mehr über unser Abreview System erfahren: https://www.abcam.com/abreviews 6.) Der Rabattcode ist nach dem Abschicken des Abreviews aktiv, und Sie können einen anderen primären Antikörper bei uns bestellen (halten Sie bei der Bestellung bitte den Rabattcode und die Bestellnummer bereit). Bitte beachten Sie, dass der Rabattcode innerhalb von 4 Monaten nach Ausstellung eingelöst werden muss. Der Rabattcode wird gültig unabhängig davon, ob Ihr Ergebnis positiv oder negativ ist. Die Bedingungen zu unserem 100% Abreview Rabatt können Sie unter dem folgenden Link nachlesen: https://www.abcam.com/collaborationdiscount Ich hoffe, diese Informationen helfen Ihnen weiter. Falls Sie einen Rabattcode erhalten möchten oder weitere Fragen haben, zögern Sie bitte nicht, sich wieder an mich zu wenden.

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Answer

Thank you for contacting us. The treatment of PFA-fixed, paraffin embedded sections (IHC-P) before incubation with priamry antibody would include antigen retrieval, blocking of endogenous peroxidases (if HRP is being used for detection), and blocking of non-specific proteins. Here are the protocol details for the respective antibodies: ab30646, ab31370, ab39636, ab59389: 1. antigen retrieval: 10 mM citrate buffer (pH6.0) boil in pressure cooker 2. block with 3% H2O2 (in fresh methanol) for 15 minutes at room temperature 3. block with 3% BSA in TBS for 30 minutes ab6672: antigen retrieval: citrate buffer pH 6 and proteinase K digestion have been tried successfully as well as no antigen retrieval. 2. block with 3% hydrogen peroxide for 10 min 3. block with 1-10% Serum for up to 1 hour ab32101: 1. antigen retrieval:10 mM Sodium Citrate Buffer, pH 6.0 in rice cooker 2. block with 3% hydrogen peroxide for 10 min 3. block with PBS + 10% serum 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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Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Rat Cell lysate - whole cell (Uterine Cell line)
Loading amount
30 µg
Specification
Uterine Cell line
Treatment
1ug/ml Prolactin
Gel Running Conditions
Reduced Denaturing (10)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 24°C

Abcam user community

Verified customer

Submitted Mar 19 2010

Application
Western blot
Sample
Human Cell lysate - whole cell (Lung)
Loading amount
10 µg
Specification
Lung
Treatment
10ng/mL IFN gamma for 24hrs
Gel Running Conditions
Reduced Denaturing
Blocking step
Milk as blocking agent for 18 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 4°C

Abcam user community

Verified customer

Submitted Jul 31 2007

Answer

Thank you for contacting us. In answer to your questions: 1. The antibody ab32101 is specific to the phosphorylated protein. We do not have another Western blot but we are confident, given the dot blot result, that this antibody will not recognize unphosphorylated JAK2. 2. We have not tested the antibody on bone marrow but our Abpromise guarantee covers this application. 3. Any carcinoma should serve well as a positive control: colon, breast, renal. 4. We do not know how often lung adenocarcinoma tissue reacts positively with this antibody. I hope this information helps. Please do not hesitate to contact us if you need any more advice or information.

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