All lanes : Anti-HNRNPA0 antibody [EP16085] (ab197023) at 1/1000 dilution

Lane 1 : Wild-type HEK293T cell lysate
Lane 2 : HNRNPA0 knockout HEK293T cell lysate
Lane 3 : A549 cell 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: 31 kDa
Observed band size: 32,34 kDa why is the actual band size different from the predicted?

Lanes 1-3: Merged signal (red and green). Green - ab197023 observed at 32,34 kDa. Red - loading control ab8245 observed at 36 kDa.

 ab197023 Anti-HNRNPA0 antibody [EP16085]  was shown to specifically react with HNRNPA0 in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266314 (knockout cell lysate ab257989) was used. Wild-type and HNRNPA0 knockout samples were subjected to SDS-PAGE. ab197023 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) 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 HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling HNRNPA0 with ab197023 at 1/1000 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/500 dilution (green). Nuclear staining on HeLa 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: ab197023 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 1/400 dilution.

Immunohistochemical analysis of paraffin-embedded Human cerebral cortex labeling HNRNPA0 with ab197023 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on Human cerebral cortex tissue 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.

Immunohistochemical analysis of paraffin-embedded Mouse spleen labeling HNRNPA0 with ab197023 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on Mouse spleen tissue 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.

Flow cytometry analysis of HeLa cells labelling HNRNPA0 (red) with purified ab197023 at dilution of 1/150. The secondary antibody used was Alexa Fluor^® 488 goat-anti-rabbit IgG (1/2000). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. Isotype control antibody was Rabbit monoclonal IgG (black). The blue line shows cells without incubation with primary antibody and secondary antibody.

All lanes : Anti-HNRNPA0 antibody [EP16085] (ab197023) at 1/10000 dilution

Lane 1 : HeLa (Human epithelial cells from cervix adenocarcinoma) lysate
Lane 2 : Jurkat (Human T cell leukemia cells from peripheral blood) lysate
Lane 3 : A549 (Human lung carcinoma) 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: 31 kDa
Observed band size: 32,34 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

Anti-HNRNPA0 antibody [EP16085] (ab197023) at 1/1000 dilution + 293 (Human embryonic kidney) lysate at 20 µg

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

Predicted band size: 31 kDa
Observed band size: 32,34 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-HNRNPA0 antibody [EP16085] (ab197023) at 1/1000 dilution

Lane 1 : Mouse brain lysate
Lane 2 : Rat brain lysate
Lane 3 : C6 (Rat glial tumor cells) lysate
Lane 4 : RAW 264.7 (Mouse macrophage cells transformed with Abelson murine leukemia virus) lysate
Lane 5 : PC-12 (Rat adrenal gland pheochromocytoma) lysate
Lane 6 : NIH/3T3 (Mouse embyro fibroblast cells) 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: 31 kDa
Observed band size: 32 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.