All lanes : Anti-HNRPAB antibody [EPR16944] (ab199724) at 1/1000 dilution

Lane 1 : Wild-type HEK293T cell lysate
Lane 2 : HNRNPAB knockout HEK293T cell lysate
Lane 3 : K-562 cell lysate
Lane 4 : Jurkat 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: 36 kDa
Observed band size: 48 kDa why is the actual band size different from the predicted?

Lanes 1-4: Merged signal (red and green). Green - ab199724 observed at 48 kDa. Red - loading control ab8245 observed at 36 kDa. 

 ab199724 Anti-HNRPAB antibody [EPR16944] was shown to specifically react with HNRPAB in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266459 (knockout cell lysate ab257467) was used.  Wild-type and HNRPAB knockout samples were subjected to SDS-PAGE.  ab199724 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at room temperature for 2. 5 hours 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 : Anti-HNRPAB antibody [EPR16944] (ab199724) at 1/5000 dilution

Lane 1 : K562 (Human chronic myelogenous leukemia cells from bone marrow) whole cell lysate
Lane 2 : Jurkat (Human T cell leukemia cells from peripheral blood) whole cell lysate
Lane 3 : HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell 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: 36 kDa
Observed band size: 42 kDa why is the actual band size different from the predicted?


Exposure time: 5 seconds

The expression profile observed is consistent with what has been described in the literature PMID: 22332140.

Blocking/Dilution buffer: 5% NFDM/TBST.

ab199724 staining HNRPAB in Jurkat (human acute T cell leukemia) cells by flow cytometry. Cells were fixed with 4% paraformaldehyde and the sample was incubated with the primary antibody at a dilution of 1/2700. A goat anti rabbit IgG (Alexa Fluor® 488) at a dilution of 1/2000 was used as the secondary antibody.

Isoytype control: Rabbit monoclonal IgG (Black)

Unlabelled control: Cell without incubation with primary antibody and secondary antibody (Blue)

Anti-HNRPAB antibody [EPR16944] (ab199724) at 1/5000 dilution + Human fetal brain lysate at 20 µg

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

Predicted band size: 36 kDa
Observed band size: 42 kDa why is the actual band size different from the predicted?


Exposure time: 10 seconds

The expression profile observed is consistent with what has been described in the literature PMID: 22332140.

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-HNRPAB antibody [EPR16944] (ab199724) at 1/1000 dilution

Lane 1 : Mouse brain lysates
Lane 2 : Rat brain lysates
Lane 3 : C6 (Rat glial tumor cells) whole cell lysate
Lane 4 : RAW 264.7 (Mouse macrophage cells transformed with Abelson murine leukemia virus) whole cell lysate
Lane 5 : PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysate
Lane 6 : 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), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 36 kDa
Observed band size: 42 kDa why is the actual band size different from the predicted?


Exposure time: 10 seconds

The expression profile observed is consistent with what has been described in the literature PMID: 22332140.

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunohistochemical analysis of paraffin-embedded Human cervix carcinoma tissue labeling HNRPAB with ab199724 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Nuclear staining on Human cervix carcinoma tissue 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).

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

Immunohistochemical analysis of paraffin-embedded mouse liver tissue labeling HNRPAB with ab199724 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Nuclear staining on mouse liver tissue 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).

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

Immunohistochemical analysis of paraffin-embedded rat cardiac muscle tissue labeling HNRPAB with ab199724 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Nuclear staining on rat cardiac muscle tissue 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).

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 Jurkat (Human T cell leukemia cells from peripheral blood) cells labeling HNRPAB with ab199724 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/500 dilution (green). Nuclear staining on Jurkat cell line is observed. The nuclear counterstain 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: ab199724 at 1/500 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.

HNRPAB was immunoprecipitated from 1mg of K562 (Human chronic myelogenous leukemia cells from bone marrow) whole cell lysate with ab199724 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab199724 at 1/1000 dilution. Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG, was used as secondary antibody at 1/1500 dilution.

Lane 1: K562 whole cell lysate 10 µg (Input). Lane 2: ab199724 IP in K562 whole cell lysate. Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab199724 in K562 whole cell lysate.

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