Anti-Hexokinase II antibody [EPR20839] - BSA and Azide free

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Hexokinase II antibody [EPR20839] - BSA and Azide free (AB228819)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab209847).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hexokinase II antibody [EPR20839] - BSA and Azide free (AB228819)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab209847).

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] - BSA and Azide free (AB228819)

Clone EPR20839 (ab228819) has been successfully conjugated by Abcam. This image was generated using Anti-Hexokinase II antibody [EPR20839] (Alexa Fluor® 647). Please refer to ab237314 for protocol details.

ab237314 staining Hexokinase II in HeLa cells. The cells were fixed with 4% formaldehyde (10 min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab237314 at 1/1000 dilution (shown in red) and ab195887, Mouse monoclonal to alpha Tubulin (Alexa Fluor^® 488), at 1/250 dilution (shown in green). Nuclear DNA was labelled with DAPI (shown in blue).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hexokinase II antibody [EPR20839] - BSA and Azide free (AB228819)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab209847).

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

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Hexokinase II antibody [EPR20839] - BSA and Azide free (AB228819)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab209847).

• IP

Supplier Data

Immunoprecipitation - Anti-Hexokinase II antibody [EPR20839] - BSA and Azide free (AB228819)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab209847).

All lanes:

Immunoprecipitation - Anti-Hexokinase II antibody [EPR20839] (<a href='/en-us/products/primary-antibodies/hexokinase-ii-antibody-epr20839-ab209847'>ab209847</a>)

Predicted band size: 102 kDa

Observed band size: 102 kDa

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• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Hexokinase II antibody [EPR20839] - BSA and Azide free (AB228819)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab209847).

• WB

Lab

Western blot - Anti-Hexokinase II antibody [EPR20839] - BSA and Azide free (AB228819)

This data was developed using the same antibody clone in a different buffer formulation (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] (<a href='/en-us/products/primary-antibodies/hexokinase-ii-antibody-epr20839-ab209847'>ab209847</a>) 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

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