Lane 1: Wild-type HAP1 cell lysate (40 µg)
Lane 2: KAT1/HAT1 knockout HAP1 cell lysate (40 µg)
Lane 3: HeLa cell lysate (20 µg)
Lane 4:Jurkat cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab194296 observed at 48 kDa. Red - loading control, ab18058, observed at 37 kDa.

ab194296 was shown to recognize KAT1/HAT1 in wild-type HAP1 cells along with additional cross-reactive bands. No band was observed when KAT1/HAT1 knockout samples were examined. Wild-type and KAT1 / HAT1 knockout samples were subjected to SDS-PAGE. Ab194296 and ab18058 (loading control to GAPDH) were diluted at 1/1000 and 1/10,000 dilution respectively and incubated overnight at 4^oC. Blots were developed with IRDye® 800CW Goat anti-Rabbit IgG (H + L) and IRDye® 680 Goat anti-Mouse IgG (H + L) secondary antibodies at 1/10,000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-KAT1 / HAT1 antibody [EPR18775] (ab194296) at 1/1000 dilution

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

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/10000 dilution

Predicted band size: 49 kDa
Observed band size: 45 kDa why is the actual band size different from the predicted?


Exposure time: 5 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-KAT1 / HAT1 antibody [EPR18775] (ab194296) at 1/1000 dilution

Lane 1 : Mouse colon lysate
Lane 2 : Rat colon lysate
Lane 3 : HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate
Lane 4 : MCF7 (Human breast adenocarcinoma cell line) whole cell lysate
Lane 5 : F9 (Mouse embyro testicular cancer cell line) whole cell lysate
Lane 6 : LLC1 (Mouse lung carcinoma cell line) whole cell lysate
Lane 7 : Mouse thymus lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 49 kDa
Observed band size: 45 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1 and 2: 3 minutes; Lane 3 and 4: 2 seconds; Lane 5, 6 and 7: 5 seconds

All lanes : Anti-KAT1 / HAT1 antibody [EPR18775] (ab194296) at 1/1000 dilution

Lane 1 : Mouse brain lysate
Lane 2 : Mouse kidney lysate
Lane 3 : Mouse spleen lysate
Lane 4 : Rat brain lysate
Lane 5 : Rat kidney lysate
Lane 6 : Rat spleen lysate
Lane 7 : C6 (Rat glial tumor cells) whole cell lysate
Lane 8 : RAW 264.7 (Mouse macrophage cells transformed with Abelson murine leukemia virus) whole cell lysate
Lane 9 : PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysate
Lane 10 : NIH/3T3 (Mouse embyro fibroblast cells) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 49 kDa
Observed band size: 45 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1, 2, 4, 5 and 6: 3 minutes; Lane 3: 1 minute; Lane 7, 8, 9 and 10: 5 seconds.

Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling KAT1 / HAT1 with ab194296 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nucleus staining on the germinal center of Human tonsil 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) (ab97051) at 1/500 dilution.

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

Immunohistochemical analysis of paraffin-embedded Human gastric adenocarcinoma tissue labeling KAT1 / HAT1 with ab194296 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nucleus staining on cancer cells of Human gastric adenocarcinoma 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) (ab97051) at 1/500 dilution.

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

Immunohistochemical analysis of paraffin-embedded mouse colon tissue labeling KAT1 / HAT1 with ab194296 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nucleus staining on epithelial cells of mouse colon 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) (ab97051) at 1/500 dilution.

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

Immunohistochemical analysis of paraffin-embedded rat colon tissue labeling KAT1 / HAT1 with ab194296 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nucleus staining on epithelial cells of rat colon 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) (ab97051) at 1/500 dilution.

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

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling KAT1 / HAT1 with ab194296 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear staining on HeLa cell line. 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 1/1000 dilution (red).
The negative controls are as follows:
-ve control 1: ab194296 at 1/500 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 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 1/1000 dilution.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized F9 (Mouse embyro testicular cancer cell line) cells labeling KAT1 / HAT1 with ab194296 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear staining on F9 cell line. 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 1/1000 dilution (red).
The negative controls are as follows:
-ve control 1: ab194296 at 1/500 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 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 1/1000 dilution.

Flow cytometric analysis of 4% paraformaldehyde-fixed HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling KAT1 / HAT1 with ab194296 at 1/150 dilution (red) compared with a rabbit monoclonal IgG isotype control (ab172730; black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (FITC) at 1/500 dilution was used as the secondary antibody.

KAT1 / HAT1 was immunoprecipitated from 1mg of HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate with ab194296 at 1/50 dilution. Western blot was performed from the immunoprecipitate using ab194296 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 10ug (Input). 

Lane 2: ab194296 IP in HeLa whole cell lysate. 

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab194296 in HeLa whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 30 seconds.

KAT1 / HAT1 was immunoprecipitated from 1mg of F9 (Mouse embyro testicular cancer cell line) whole cell lysate with ab194296 at 1/50 dilution. Western blot was performed from the immunoprecipitate using ab194296 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1: F9 whole cell lysate 10ug (Input). 

Lane 2: ab194296 IP in F9 whole cell lysate. 

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab194296 in F9 whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 30 seconds.