Anti-Hexokinase II antibody [EPR20839]

• WB

Lab

Western blot - Anti-Hexokinase II antibody [EPR20839] (AB209847)

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 wild-type HCT 116 cells in western blot with loss of signal observed in HK2 knockout cell line ab273721 (HK2 knockout cell lysate ab275250). Wild-type and HK2 knockout HCT 116 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.

All lanes:

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

Lane 1:

Wild-type HCT116 cell lysate at 20 µg

Lane 2:

HK2 knockout HCT116 cell lysate at 20 µg

Lane 2:

Western blot - Human HK2 (Hexokinase II) knockout HCT116 cell line (<a href='/en-us/products/cell-lines/human-hk2-hexokinase-ii-knockout-hct116-cell-line-ab273721'>ab273721</a>)

Lane 3:

HeLa cell lysate at 20 µg

Lane 4:

SH-SY5Y cell lysate at 20 µg

Predicted band size: 102 kDa

Observed band size: 105 kDa

false

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hexokinase II antibody [EPR20839] (AB209847)

Immunohistochemical analysis of formalin fixed paraffin embedded human invasive colon carcinoma labelling hexokinase II with ab209847 at a concentration of 0.5µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins. ab209847 Anti-Hexokinase II antibody [EPR20839] was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual).

• IHC-P

Supplier Data

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.

• IHC-P

Supplier Data

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.

• ICC/IF

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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.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Hexokinase II antibody [EPR20839] (AB209847)

Intracellular 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.

• IP

Supplier Data

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.

All lanes:

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

Predicted band size: 102 kDa

Observed band size: 102 kDa

false

• ICC/IF

Supplier Data

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.

• WB

Supplier Data

Western blot - Anti-Hexokinase II antibody [EPR20839] (AB209847)

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.

All lanes:

Western blot - 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, 2, 3, 4, 5 and 8:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Lanes 6 - 7:

Western blot - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/4000 dilution

Predicted band size: 102 kDa

Observed band size: 102 kDa

true

• WB

Supplier Data

Western blot - Anti-Hexokinase II antibody [EPR20839] (AB209847)

Blocking/Dilution buffer : 5% NFDM/TBST.

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

All lanes:

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

Lane 1:

HeLa (human epithelial cell line from cervix adenocarcinoma), whole cell lysate at 10 µg

Lane 2:

HKDC1-knockdown HeLa (human epithelial cell line from cervix adenocarcinoma) cell lysate at 10 µg

Lane 3:

Hexokinase I-knockdown HeLa (human epithelial cell line from cervix adenocarcinoma) cell lysate at 10 µg

Lane 4:

Hexokinase II-knockdown HeLa (human epithelial cell line from cervix adenocarcinoma) cell lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 102 kDa

Observed band size: 102 kDa

true

Exposure time: 41s

• WB

Lab

Western blot - Anti-Hexokinase II antibody [EPR20839] (AB209847)

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.

All lanes:

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

Lane 1:

Wild-type HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate at 20 µg

Lane 2:

HK2 knockout HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate at 20 µg

Lane 2:

Western blot - Human HK2 (Hexokinase II) knockout HEK-293 cell line (<a href='/en-us/products/cell-lines/human-hk2-hexokinase-ii-knockout-hek-293-cell-line-ab269485'>ab269485</a>)

Lane 3:

HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

Lane 4:

A549 (Human lung carcinoma cell line) whole cell lysate at 20 µg

Predicted band size: 102 kDa

Observed band size: 110 kDa

false

• Dot

Supplier Data

Dot Blot - Anti-Hexokinase II antibody [EPR20839] (AB209847)

Dot blot analysis of Hexokinase Type III/HK3 using ab321996 at 1 : 1000 (0.525 ug/ml) followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1 : 100,000 dilution.

Lane1 : His-tagged human HK3 fragment
Lane2 : His-tagged human HK2 fragment
Lane3 : His-tagged human HK1 fragment
Lane4 : His-tagged human HKDC1 fragment

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

This antibody does not cross-react with human HK1, HK2 and HKDC1.

All lanes:

Dot Blot - Anti-Hexokinase Type III/HK3 antibody [EPR29196-29] (<a href='/en-us/products/primary-antibodies/hexokinase-type-iii-hk3-antibody-epr29196-29-ab321996'>ab321996</a>) at 1/1000 dilution

Lane 1:

His-tagged human HK3 fragment at 5 ng

Lane 2:

His-tagged human HK2 fragment at 5 ng

Lane 3:

His-tagged human HK1 fragment at 5 ng

Lane 4:

His-tagged human HKDC1 fragment at 5 ng

Secondary

All lanes:

Dot Blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-Hexokinase II antibody [EPR20839] (AB209847)

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

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

All lanes:

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

Lane 1:

HKDC1 recombinant protein (aa16-458) at 0.01 µg

Lane 2:

Hexokinase II recombinant protein (aa16-458) at 0.01 µg

Lane 3:

Hexokinase I recombinant protein (aa16-458) at 0.01 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 102 kDa

Observed band size: 50 kDa

true

Exposure time: 3min