RecombinantRabMAb

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

Reviews (4)Q&A (6)References (135)
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-JAK2 (phospho Y1007 + Y1008) antibody [E132] (ab32101)
  • Western blot - Anti-JAK2 (phospho Y1007 + Y1008) antibody [E132] (ab32101)
  • Western blot - Anti-JAK2 (phospho Y1007 + Y1008) antibody [E132] (ab32101)
  • Western blot - Anti-JAK2 (phospho Y1007 + Y1008) antibody [E132] (ab32101)
  • Immunocytochemistry/ Immunofluorescence - Anti-JAK2 (phospho Y1007 + Y1008) antibody [E132] (ab32101)
  • Dot Blot - Anti-JAK2 (phospho Y1007 + Y1008) antibody [E132] (ab32101)
  • Western blot - Anti-JAK2 (phospho Y1007 + Y1008) antibody [E132] (ab32101)
  • Flow Cytometry (Intracellular) - Anti-JAK2 (phospho Y1007 + Y1008) antibody [E132] (ab32101)
  • ELISA - Anti-JAK2 (phospho Y1007 + Y1008) antibody [E132] (ab32101)
  • ELISA - Anti-JAK2 (phospho Y1007 + Y1008) antibody [E132] (ab32101)
  • Western blot - Anti-JAK2 (phospho Y1007 + Y1008) antibody [E132] (ab32101)
  • Anti-JAK2 (phospho Y1007 + Y1008) antibody [E132] (ab32101)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [E132] to JAK2 (phospho Y1007 + Y1008)
  • Suitable for: ELISA, Flow Cyt (Intra), WB, ICC/IF, IHC-P, Dot blot
  • Reacts with: Mouse, Rat, Human

Conjugates logo Related conjugates and formulations

Alexa Fluor® 488 Alexa Fluor® 594 Alexa Fluor® 647 Carrier Free

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.

    The mouse and rat recommendation is based on the WB results. We do not guarantee IHC-P for mouse and rat.

  • Tested applications

    Suitable for: ELISA, Flow Cyt (Intra), WB, ICC/IF, IHC-P, Dot blotmore details

  • Species reactivity

    Reacts with: Mouse, Rat, Human

  • Immunogen

    Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.

  • Positive control

    • WB: Hepa1-6, MEF, C6, Jurkat treated with Pervanadate and Jurkat cell lysates. IHC-P: Human differentiated squamous cell carcinoma tissue. ICC/IF: Jurkat cells (treated with Pervanadate). Flow Cyt (intra): Jurkat starved of serum for 16 hours then treated with 1mM Pervanadate for 30 minutes.

  • General notes

    This product is a recombinant monoclonal antibody, which offers several advantages including:

    • - High batch-to-batch consistency and reproducibility
    • - Improved sensitivity and specificity
    • - Long-term security of supply
    • - Animal-free production
    For more information see here.

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

Properties

Applications

The Abpromise guarantee

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.
Flow Cyt (Intra)
1/20.
WB (3)
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)
ICC/IF (1)
1/1000.
IHC-P
Use at an assay dependent concentration. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

The mouse and rat recommendation is based on the WB results. We do not guarantee IHC-P for mouse and rat.
Dot blot
1/1000.
Notes
ELISA
Use at an assay dependent concentration.
Flow Cyt (Intra)
1/20.
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)
ICC/IF
1/1000.
IHC-P
Use at an assay dependent concentration. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

The mouse and rat recommendation is based on the WB results. We do not guarantee IHC-P for mouse and rat.
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

    see all

  • 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

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-JAK2 (phospho Y1007 + Y1008) antibody [E132] (ab32101)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-JAK2 (phospho Y1007 + Y1008) antibody [E132] (ab32101)

    ab32101 showing positive staining in Human differentiated squamous cell carcinoma of the cervix tissue at 1/10000 dilution. Goat Anti-Rabbit IgG H&L (HRP) was used as secondary antibody. Antigen retreival was carried out by Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

    Nuclear staining on human differentiated squamous cell carcinoma of the cervix without alkaline phosphatase treatment (image A). No staining on human differentiated squamous cell carcinoma of the cervix with alkaline phosphatase treatment (image B)

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

    Lane 1 : Hepa1-6 (Mouse hepatoma epithelial cell) whole cell lysate
    Lane 2 : Hepa1-6 (Mouse hepatoma epithelial cell) treated with 100 µM pervanadate for 30 minutes whole cell lysate
    Lane 3 : MEF (Mouse embryonic fibroblast (immortalized)) whole cell lysate
    Lane 4 : MEF (Mouse embryonic fibroblast (immortalized)) treated with 100 µM pervanadate for 30 minutes whole cell lysate

    Lysates/proteins at 15 µg per lane.

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

    Predicted band size: 130 kDa



    The extra bands are undefined.

  • Western blot - Anti-JAK2 (phospho Y1007 + Y1008) antibody [E132] (ab32101)
    Western blot - Anti-JAK2 (phospho Y1007 + Y1008) antibody [E132] (ab32101)
    All lanes : Anti-JAK2 (phospho Y1007 + Y1008) antibody [E132] (ab32101)

    Lane 1 : Mouse hippocampus lysate
    Lane 2 : Mouse P240 hippocampus lysate
    Lane 3 : Mouse P7 hippocampus lysate
    Lane 4 : Rat hippocampus lysate
    Lane 5 : Rat P7 hippocampus lysate
    Lane 6 : Rat brain cortex lysate
    Lane 7 : Human brain lysate
    Lane 8 : Mouse brain lysate
    Lane 9 : Rat brain lysate
    Lane 10 : C6 (Rat glial tumor glial cell) whole cell lysate
    Lane 11 : C6 (Rat glial tumor glial cell) treated with 50mM pervanadate for 5 minutes whole cell lysate

    Lysates/proteins at 15 µg per lane.

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

    Predicted band size: 130 kDa

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

  • Immunocytochemistry/ Immunofluorescence - Anti-JAK2 (phospho Y1007 + Y1008) antibody [E132] (ab32101)
    Immunocytochemistry/ Immunofluorescence - Anti-JAK2 (phospho Y1007 + Y1008) antibody [E132] (ab32101)

    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).

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

    Dot blot analysis of human 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.

    Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) was used as the secondary antibody at a dilution of 1/20,000.

    Blocking/Dilution buffer: 5% NFDM/TBST.

  • Western blot - Anti-JAK2 (phospho Y1007 + Y1008) antibody [E132] (ab32101)
    Western blot - Anti-JAK2 (phospho Y1007 + Y1008) antibody [E132] (ab32101)
    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 (Intracellular) - Anti-JAK2 (phospho Y1007 + Y1008) antibody [E132] (ab32101)
    Flow Cytometry (Intracellular) - Anti-JAK2 (phospho Y1007 + Y1008) antibody [E132] (ab32101)

    Intracellular 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 Fluorr®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. Unstimulated Jurkat cells were used as a negative control (Green). 

     

     

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

    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.

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

    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.

     

  • Western blot - Anti-JAK2 (phospho Y1007 + Y1008) antibody [E132] (ab32101)
    Western blot - Anti-JAK2 (phospho Y1007 + Y1008) antibody [E132] (ab32101)This image is courtesy of an anonymous Abreview
    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

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

Protocols

To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.

Click here to view the general protocols

References (135)

Publishing research using ab32101? Please let us know so that we can cite the reference in this datasheet.

ab32101 has been referenced in 135 publications.

  • Bian G  et al. DGT, a novel heterocyclic diterpenoid, effectively suppresses psoriasis via inhibition of STAT3 phosphorylation. Br J Pharmacol 178:636-653 (2021). PubMed: 33140855
  • Li Y  et al. Potential effect of Maxing Shigan decoction against coronavirus disease 2019 (COVID-19) revealed by network pharmacology and experimental verification. J Ethnopharmacol 271:113854 (2021). PubMed: 33513419
  • Qu L  et al. Fraxetin Inhibits the Proliferation and Metastasis of Glioma Cells by Inactivating JAK2/STAT3 Signaling. Evid Based Complement Alternat Med 2021:5540139 (2021). PubMed: 33959183
  • Du T  et al. MiR-138-1-3p alters the stemness and radiosensitivity of tumor cells by targeting CRIPTO and the JAK2/STAT3 pathway in nasopharyngeal carcinoma. Ann Transl Med 9:485 (2021). PubMed: 33850882
  • Jin Y  et al. Irradiation-Induced Activated Microglia Affect Brain Metastatic Colonization of NSCLC Cells via miR-9/CDH1 Axis. Onco Targets Ther 14:1911-1922 (2021). PubMed: 33758511
View all Publications for this product

Customer reviews and Q&As

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Immunocytochemistry/ Immunofluorescence abreview for Anti-JAK2 (phospho Y1007 + Y1008) antibody [E132]

 Good
Abreviews
abreview image
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (C42B Prostate cancer cell line)
Permeabilization
Yes - 0.1% Triton X-100
Specification
C42B Prostate cancer cell line
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C
Fixative
Paraformaldehyde
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Abcam user community

Verified customer

Submitted Feb 12 2020

Western blot abreview for Anti-JAK2 (phospho Y1007 + Y1008) antibody [E132]

 Poor
Abreviews
abreview image
Application
Western blot
Sample
Human Cell lysate - whole cell (DU145 Prostate cancer cell line)
Gel Running Conditions
Reduced Denaturing
Loading amount
20 µg
Specification
DU145 Prostate cancer cell line
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C
Read More

Abcam user community

Verified customer

Submitted Feb 05 2020

Western blot abreview for Anti-JAK2 (phospho Y1007 + Y1008) antibody [E132]

 Excellent
Abreviews
abreview image
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
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Abcam user community

Verified customer

Submitted Mar 19 2010

Western blot abreview for Anti-JAK2 (phospho Y1007 + Y1008) antibody [E132]

 Poor
Abreviews
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
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Abcam user community

Verified customer

Submitted Jul 31 2007

Question

I purchased 12 antibodies from your company.
Some didn't work and I need your help.

I used mouse liver tissue. I attached our lab's protocol.
Apply 25ug of protein in each well.
The gel I used is NuPAGE 4-12% Bis-Tris Gel.
I tried 1:1000 dilution of JAK2 and 1:5000 of phospho JAK2 in 5%NFDM TBST.
Both didn't work. No bands, even with 2 hour exposure.

Please give me your advice.

Read More
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/JAK2-phospho-Y1007--Y1008-antibody-ab68268.html).

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Question

What is the conc of lot GR18831-6 (ab32101)

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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!
https://www.abcam.com/abreviews

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Question

I am very interested in purchasing JAK2 (phospho Y1007 + Y1008)
antibody [E132] (ab32101) , but I would know whic positive control
(name of cell line, type and time of treatment) is used in western
blot image and moreover if this lysate is available by abcam,

thanks.

Read More
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

Haben Sie Erfahrungen zur Verwendung dieses AKs an Gefrierschnitten?

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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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I have a question regarding the treatment of tissue prior to applying the primary antibody. Do you recommend microwave? Enzymatic? The tissue I have in mind is human salivary gland. I'm planning to use ab6672, ab59389, ab39636, ab32101, ab31370 and ab30646. Different recommendations?

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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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Dear Sirs, I am potentially very interested in purchasing this mAb but I would like first to know: 1) whether this mAb is specific for the Y1007-Y1008 phosphorylated form of JAK2 or also detects the non-phosphorylated form (could you please provide a western blot, as the one shown in the website is not informative in this regard?) 2) whether you had the chance to test this mAb also on formalin-fixed paraffin-embedded decalcified bone marrow biopsies; in fact, the additional decalcification step required by bone material often denatures epitopes otherwise resistant to formalin fixation and paraffin embedding 3) which tissues can be used as positive controls, besides lung adenocarcinoma 4) what is the frequency of positivity of lung adenocarcinoma ?

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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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