All lanes : Anti-ketohexokinase antibody [EPR15847] (ab197593) at 1/1000 dilution

Lane 1 : Wild-type HEK293T cell lysate
Lane 2 : KHK knockout HEK293T cell lysate
Lane 3 : Human kidney tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution

Predicted band size: 33 kDa
Observed band size: 33 kDa

Lanes 1-3: Merged signal (red and green). Green - ab197593 observed at 33 kDa. Red - loading control ab7291 observed at 50 kDa.

 ab197593 Anti-ketohexokinase antibody [EPR15847] was shown to specifically react with ketohexokinase in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab267292 (knockout cell lysate ab258017) was used. Wild-type and ketohexokinase knockout samples were subjected to SDS-PAGE. ab197593 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (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 HepG2 (Human liver hepatocellular carcinoma) cells labeling ketohexokinase with ab197593 at 1/1000 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/500 dilution (green). Cytoplasm and nuclear staining on HepG2 cell line is observed. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).
The negative controls are as follows:
-ve control 1: ab197593 at 1/1000 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
-ve control 2: ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/500 dilution.

 

All lanes : Anti-ketohexokinase antibody [EPR15847] (ab197593) at 1/5000 dilution

Lane 1 : Human fetal kidney lysate
Lane 2 : Human fetal liver lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 33 kDa
Observed band size: 33 kDa

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-ketohexokinase antibody [EPR15847] (ab197593) at 1/2500 dilution

Lane 1 : Mouse kidney lysate
Lane 2 : Mouse liver lysate
Lane 3 : Rat kidney lysate
Lane 4 : Rat liver lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 33 kDa
Observed band size: 33 kDa

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunohistochemical analysis of paraffin-embedded Human liver tissue labeling ketohexokinase with ab197593 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Cytoplasm and nuclear staining on Human liver tissues is observed. Counter stained with Hematoxylin.

Negative control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

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

Ketohexokinase was immunoprecipitated from Human fetal kidney lysate with ab197593 at 1/100 dilution. Western blot was performed from the immunoprecipitate using ab197593 at 1/2000 dilution. Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG, was used as secondary antibody at 1/1500 dilution.

Lane 1: Human fetal kidney lysate 10 µg (Input). Lane 2: ab197593 IP in Human fetal kidney lysate. Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab197593 in Human fetal kidney lysate.

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