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.

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.

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 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.

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.

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.

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.

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.

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.

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.

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.