Validated using a knockout cell lineRecombinantRabMAb

Recombinant Anti-Hexokinase II antibody [EPR20839] (ab209847)

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Western blot - Anti-Hexokinase II antibody [EPR20839] (ab209847)
  • Immunocytochemistry/ Immunofluorescence - Anti-Hexokinase II antibody [EPR20839] (ab209847)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hexokinase II antibody [EPR20839] (ab209847)
  • Flow Cytometry - Anti-Hexokinase II antibody [EPR20839] (ab209847)
  • Western blot - Anti-Hexokinase II antibody [EPR20839] (ab209847)
  • Western blot - Anti-Hexokinase II antibody [EPR20839] (ab209847)
  • Immunoprecipitation - Anti-Hexokinase II antibody [EPR20839] (ab209847)
  • Immunocytochemistry/ Immunofluorescence - Anti-Hexokinase II antibody [EPR20839] (ab209847)
  • Western blot - Anti-Hexokinase II antibody [EPR20839] (ab209847)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hexokinase II antibody [EPR20839] (ab209847)
  • Western blot - Anti-Hexokinase II antibody [EPR20839] (ab209847)
  • Anti-Hexokinase II antibody [EPR20839] (ab209847)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EPR20839] to Hexokinase II
  • Suitable for: WB, IHC-P, ICC/IF, IP, Flow Cyt
  • Knockout validated
  • Reacts with: Mouse, Rat, Human, Recombinant fragment

Overview

  • Product name

    Anti-Hexokinase II antibody [EPR20839]
    See all Hexokinase II primary antibodies

  • Description

    Rabbit monoclonal [EPR20839] to Hexokinase II

  • Host species

    Rabbit

  • Specificity

    Not recommended for rat and mouse tissues in IHC.

  • Tested applications

    Suitable for: WB, IHC-P, ICC/IF, IP, Flow Cytmore details

  • Species reactivity

    Reacts with: Mouse, Rat, Human, Recombinant fragment

  • Immunogen

    Recombinant fragment. This information is proprietary to Abcam and/or its suppliers.

  • Positive control

    • WB: Hexokinase II recombinant protein (aa16-458); HEK-293, HCT116, HeLa, SH-SY5Y and NIH/3T3 whole cell lysates; HKDC1 and Hexokinase I-knockdown HeLa cell lysates; Rat testis and muscle lysates; Human testis, colon and fetal heart lysates; Mouse muscle lysate. IHC-P: Human heart and pancreatic adenocarcinoma tissues. ICC/IF: HeLa and NIH/3T3 cells. Flow Cyt: HeLa cells. IP: HeLa whole cell lysate.

  • 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 ab209847 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
WB (2)
1/1000. Detects a band of approximately 102 kDa (predicted molecular weight: 102 kDa).
IHC-P
1/500. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Not recommended for rat and mouse tissues.
ICC/IF
1/100.
IP
1/30.
Flow Cyt
1/60.
Notes
WB
1/1000. Detects a band of approximately 102 kDa (predicted molecular weight: 102 kDa).
IHC-P
1/500. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Not recommended for rat and mouse tissues.
ICC/IF
1/100.
IP
1/30.
Flow Cyt
1/60.

Target

  • Tissue specificity

    Predominant hexokinase isozyme expressed in insulin-responsive tissues such as skeletal muscle.

  • Pathway

    Carbohydrate metabolism; hexose metabolism.

  • Sequence similarities

    Belongs to the hexokinase family.
    Contains 2 hexokinase domains.

  • Domain

    The N- and C-terminal halves of this hexokinase show extensive sequence similarity to each other. The catalytic activity is associated with the C-terminus while regulatory function is associated with the N-terminus. Each domain can bind a single glucose and Gluc-6-P molecule.

  • Cellular localization

    Mitochondrion outer membrane. Its hydrophobic N-terminal sequence may be involved in membrane binding.
  • Information by UniProt

  • Database links

    see all

  • Alternative names

    • DKFZp686M1669 antibody
    • Hexokinase 2 antibody
    • Hexokinase 2 muscle antibody
    • Hexokinase type II antibody
    • Hexokinase-2 antibody
    • HK 2 antibody
    • HK II antibody
    • HK2 antibody
    • HKII antibody
    • HxK 2 antibody
    • HxK2 antibody
    • HXK2_HUMAN antibody
    • Muscle form hexokinase antibody
    see all

Images

  • Western blot - Anti-Hexokinase II antibody [EPR20839] (ab209847)
    Western blot - Anti-Hexokinase II antibody [EPR20839] (ab209847)
    All lanes : Anti-Hexokinase II antibody [EPR20839] (ab209847) at 1/1000 dilution

    Lane 1 : Wild-type HCT116 cell lysate
    Lane 2 : HK2 knockout HCT116 cell lysate
    Lane 3 : HeLa cell lysate
    Lane 4 : SH-SY5Y cell lysate

    Lysates/proteins at 20 µg per lane.

    Performed under reducing conditions.

    Predicted band size: 102 kDa
    Observed band size: 105 kDa why is the actual band size different from the predicted?



    Lanes 1 - 4: Merged signal (red and green). Green - ab209847 observed at 105 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

    ab209847 was shown to react with Anti-Hexokinase II in HCT 116 wild-type cells in western blot with loss of signal observed in HK2 knockout cell line ab273721 (HK2 knockout cell lysate ab275250). HCT 116 wild-type and HK2 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween®) before incubation with ab209847 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

  • Immunocytochemistry/ Immunofluorescence - Anti-Hexokinase II antibody [EPR20839] (ab209847)
    Immunocytochemistry/ Immunofluorescence - Anti-Hexokinase II antibody [EPR20839] (ab209847)

    Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (human cervix adenocarcinoma epithelial cell) cells labeling Hexokinase II with ab209847 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Cytoplasmic staining in HeLa cells.

    The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) (ab195889) (red) at 1/200 dilution.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hexokinase II antibody [EPR20839] (ab209847)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hexokinase II antibody [EPR20839] (ab209847)

    Immunohistochemical analysis of paraffin-embedded human pancreatic adenocarcinoma tissue labeling Hexokinase II with ab209847 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Strong cytoplasmic staining in human pancreatic adenocarcinoma (PMID: 26137268) is observed. Counter stained with Hematoxylin.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

    Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

  • Flow Cytometry - Anti-Hexokinase II antibody [EPR20839] (ab209847)
    Flow Cytometry - Anti-Hexokinase II antibody [EPR20839] (ab209847)

    Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized HeLa (human epithelial cell line from cervix adenocarcinoma) cell line labeling Hexokinase II with ab209847 at 1/60 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue).Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) at 1/2000 dilution was used as the secondary antibody.

  • Western blot - Anti-Hexokinase II antibody [EPR20839] (ab209847)
    Western blot - Anti-Hexokinase II antibody [EPR20839] (ab209847)
    All lanes : Anti-Hexokinase II antibody [EPR20839] (ab209847) at 1/1000 dilution

    Lane 1 : Wild-type HEK-293 cell lysate
    Lane 2 : HK2 knockout HEK-293 cell lysate
    Lane 3 : HeLa cell lysate
    Lane 4 : A549 cell lysate

    Lysates/proteins at 20 µg per lane.

    Performed under reducing conditions.

    Predicted band size: 102 kDa
    Observed band size: 110 kDa why is the actual band size different from the predicted?



    Lanes 1 - 4: Merged signal (red and green). Green - ab209847 observed at 110 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

    ab209847 was shown to react with Hexokinase II in wild-type HEK-293 cells in western blot with loss of signal observed in HK2 knockout sample. Wild-type and HK2 knockout HEK-293  cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween®) before incubation with ab209847 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 1000 Dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

  • Western blot - Anti-Hexokinase II antibody [EPR20839] (ab209847)
    Western blot - Anti-Hexokinase II antibody [EPR20839] (ab209847)
    All lanes : Anti-Hexokinase II antibody [EPR20839] (ab209847) at 1/1000 dilution

    Lane 1 : HeLa (human epithelial cell line from cervix adenocarcinoma), whole cell lysate
    Lane 2 : HKDC1-knockdown HeLa (human epithelial cell line from cervix adenocarcinoma) cell lysate
    Lane 3 : Hexokinase I-knockdown HeLa (human epithelial cell line from cervix adenocarcinoma) cell lysate
    Lane 4 : Hexokinase II-knockdown HeLa (human epithelial cell line from cervix adenocarcinoma) cell lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

    Developed using the ECL technique.

    Predicted band size: 102 kDa
    Observed band size: 102 kDa


    Exposure time: 41 seconds


    Blocking/Dilution buffer: 5% NFDM/TBST.

    The blot was developed on a BIO-RAD® ChemiDoc™ MP instrument.

  • Immunoprecipitation - Anti-Hexokinase II antibody [EPR20839] (ab209847)
    Immunoprecipitation - Anti-Hexokinase II antibody [EPR20839] (ab209847)

    Hexokinase II was immunoprecipitated from 0.35 mg HeLa (humanepithelial cell line from cervix adenocarcinoma) whole cell lysate with ab209847 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab209847 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

    Lane 1: HeLa whole cell lysate 10 μg (Input).
    Lane 2: ab209847 IP in HeLa whole cell lysate.
    Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab209847 in HeLa whole cell lysate.

    Exposure time: 30 seconds.

    Blocking and dilution buffer and concentration: 5% NFDM/TBST.

  • Immunocytochemistry/ Immunofluorescence - Anti-Hexokinase II antibody [EPR20839] (ab209847)
    Immunocytochemistry/ Immunofluorescence - Anti-Hexokinase II antibody [EPR20839] (ab209847)

    Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 (mouse embryonic fibroblast cell line) cells labeling Hexokinase II with ab209847 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Cytoplasmic staining in NIH/3T3 cells. 

    The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) (ab195889) (red) at 1/200 dilution.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution.

  • Western blot - Anti-Hexokinase II antibody [EPR20839] (ab209847)
    Western blot - Anti-Hexokinase II antibody [EPR20839] (ab209847)
    All lanes : Anti-Hexokinase II antibody [EPR20839] (ab209847) at 1/1000 dilution

    Lane 1 : Rat testis lysate at 20 µg
    Lane 2 : Rat muscle lysate at 20 µg
    Lane 3 : SH-SY5Y (human neuroblastoma cell line from bone marrow), whole cell lysate at 20 µg
    Lane 4 : Human testis lysate at 20 µg
    Lane 5 : Mouse muscle lysate at 20 µg
    Lane 6 : Human colon lysate at 10 µg
    Lane 7 : Human fetal heart lysate at 10 µg
    Lane 8 : NIH/3T3 (mouse embryonic fibroblast cell line), whole cell lysate at 10 µg

    Secondary
    Lanes 1-5 & 8 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution
    Lanes 6-7 : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/4000 dilution

    Developed using the ECL technique.

    Predicted band size: 102 kDa
    Observed band size: 102 kDa



     

    Exposure times: Lanes 1-3,6,7: 3 minutes; Lanes 4,5,8: 58 seconds.

    Blocking/Dilution buffer: 5% NFDM/TBST.

    The blot was developed on a BIO-RAD® ChemiDoc™ MP instrument.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hexokinase II antibody [EPR20839] (ab209847)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hexokinase II antibody [EPR20839] (ab209847)

    Immunohistochemical analysis of paraffin-embedded human heart tissue labeling Hexokinase II with ab209847 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Granularly cytoplasmic staining in human heart (PMID: 4058069; PMID: 26722360) is observed. Counter stained with Hematoxylin.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

    Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

  • Western blot - Anti-Hexokinase II antibody [EPR20839] (ab209847)
    Western blot - Anti-Hexokinase II antibody [EPR20839] (ab209847)
    All lanes : Anti-Hexokinase II antibody [EPR20839] (ab209847) at 1/10000 dilution

    Lane 1 : HKDC1 recombinant protein (aa16-458)
    Lane 2 : Hexokinase II recombinant protein (aa16-458)
    Lane 3 : Hexokinase I recombinant protein (aa16-458)

    Lysates/proteins at 0.01 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

    Developed using the ECL technique.

    Predicted band size: 102 kDa
    Observed band size: 50 kDa why is the actual band size different from the predicted?


    Exposure time: 3 minutes


    Blocking/dilution buffer and concentration: 5% NFDM/TBST.

    The blot was developed on a BIO-RAD® ChemiDoc™ MP instrument.

  • Anti-Hexokinase II antibody [EPR20839] (ab209847)
    Anti-Hexokinase II antibody [EPR20839] (ab209847)

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 (14)

Publishing research using ab209847? Please let us know so that we can cite the reference in this datasheet.

ab209847 has been referenced in 14 publications.

  • Tan X  et al. Knockdown of lncRNA NEAT1 suppresses hypoxia-induced migration, invasion and glycolysis in anaplastic thyroid carcinoma cells through regulation of miR-206 and miR-599. Cancer Cell Int 20:132 (2020). PubMed: 32336952
  • Guan Y  et al. Circular RNA circPITX1 knockdown inhibits glycolysis to enhance radiosensitivity of glioma cells by miR-329-3p/NEK2 axis. Cancer Cell Int 20:80 (2020). PubMed: 32190004
  • Tian S  et al. Circ-FOXM1 contributes to cell proliferation, invasion, and glycolysis and represses apoptosis in melanoma by regulating miR-143-3p/FLOT2 axis. World J Surg Oncol 18:56 (2020). PubMed: 32183822
  • Men L  et al. Cardiac Transcriptome Analysis Reveals Nr4a1 Mediated Glucose Metabolism Dysregulation in Response to High-Fat Diet. Genes (Basel) 11:N/A (2020). PubMed: 32610475
  • Yang D  et al. Oxidized ATM promotes breast cancer stem cell enrichment through energy metabolism reprogram-mediated acetyl-CoA accumulation. Cell Death Dis 11:508 (2020). PubMed: 32641713
View all Publications for this product

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1-2 of 2 Abreviews

Western blot abreview for Anti-Hexokinase II antibody [EPR20839]

 Good
Abreviews
abreview image
Application
Western blot
Sample
Mouse Tissue lysate - whole (brain)
Gel Running Conditions
Reduced Denaturing (TEO TRICINE 4-20% gel (ab119209))
Loading amount
20 µg
Specification
brain
Blocking step
BSA as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Read More

Dr. Yonatan Feuermann

Verified customer

Submitted Apr 12 2021

Western blot abreview for Anti-Hexokinase II antibody [EPR20839]

 Excellent
Abreviews
abreview image
Application
Western blot
Sample
Human Cell lysate - whole cell (Induced pluripotent stem cells (iPSCs))
Gel Running Conditions
Reduced Denaturing (10%)
Loading amount
25 µg
Specification
Induced pluripotent stem cells (iPSCs)
Blocking step
Milk as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 23°C
Read More

Herr Dr. Vladimir Milenkovic

Verified customer

Submitted Jun 30 2020

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