重组Anti-ketohexokinase抗体[EPR15847]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ketohexokinase antibody [EPR15847] (AB197593)

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.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-ketohexokinase antibody [EPR15847] (AB197593)

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.

• IP

Supplier Data

Immunoprecipitation - Anti-ketohexokinase antibody [EPR15847] (AB197593)

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.

All lanes:

Immunoprecipitation - Anti-ketohexokinase antibody [EPR15847] (ab197593)

Predicted band size: 33 kDa

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• WB

Lab

Western blot - Anti-ketohexokinase antibody [EPR15847] (AB197593)

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.

All lanes:

Western blot - Anti-ketohexokinase antibody [EPR15847] (ab197593) at 1/1000 dilution

Lane 1:

Wild-type HEK293T cell lysate at 20 µg

Lane 2:

Western blot - Human KHK (ketohexokinase) knockout HEK-293T cell lysate (<a href='/products/cell-lysates/human-khk-ketohexokinase-knockout-hek-293t-cell-lysate-ab258017'>ab258017</a>) at 20 µg

Lane 3:

Human kidney tissue lysate at 20 µg

Secondary

Lanes 1 - 3:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/20000 dilution

Lanes 1 - 3:

Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (<a href='/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/20000 dilution

Predicted band size: 33 kDa

Observed band size: 50 kDa,33 kDa

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• WB

Supplier Data

Western blot - Anti-ketohexokinase antibody [EPR15847] (AB197593)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-ketohexokinase antibody [EPR15847] (ab197593) at 1/5000 dilution

Lane 1:

Human fetal kidney lysate at 20 µg

Lane 2:

Human fetal liver lysate at 20 µg

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

false

• WB

Supplier Data

Western blot - Anti-ketohexokinase antibody [EPR15847] (AB197593)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-ketohexokinase antibody [EPR15847] (ab197593) at 1/2500 dilution

Lane 1:

Mouse kidney lysate at 10 µg

Lane 2:

Mouse liver lysate at 10 µg

Lane 3:

Rat kidney lysate at 10 µg

Lane 4:

Rat liver lysate at 10 µg

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

false