All lanes : Anti-NFIB / NF1B2 + NFIC/CTF antibody [EPR14504] (ab200829) at 1/10000 dilution

Lane 1 : HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate
Lane 2 : MCF7 (Human breast adenocarcinoma cell line) 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: 56, 47 kDa
Observed band size: 45-65 kDa why is the actual band size different from the predicted?


Exposure time: 30 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

Based on the sequence analysis, ab200829 recognizes six isoforms within human with predicted Mw’s of 56KDa, 55KDa, 48KDa, 45KDa, 48KDa and 49KDa, respectively.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling NFIB / NF1B2 + NFIC with ab200829 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 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: ab200829 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.

Immunohistochemical analysis of paraffin-embedded human cervix carcinoma tissue labeling NFIB / NF1B2 + NFIC with ab200829 at 1/1000 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) 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) at 1/500 dilution.

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

Flow cytometric analysis of 2% paraformaldehyde-fixed HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling NFIB / NF1B2 + NFIC with ab200829 at 1/200 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 1/150 dilution was used as the secondary antibody.

NFIB/NF1B2 + NFIC was immunoprecipitated from 1mg of HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate with ab200829 at 1/70 dilution. Western blot was performed from the immunoprecipitate using ab200829 at 1/5000 dilution. Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG, was used as secondary antibody at 1/1500 dilution.

Lane 1: HeLa whole cell lysate, 10 µg (Input).

Lane 2: ab200829 IP in HeLa whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab200829 in HeLa whole cell lysate.
Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 10 seconds.

Immunohistochemical analysis of paraffin-embedded Human breast carcinoma tissue labeling NFIB / NF1B2 + NFIC with ab200829 at 1/1000 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on Human breast 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) 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 cardiac muscle tissue labeling NFIB / NF1B2 + NFIC with ab200829 at 1/1000 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on mouse 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) at 1/500 dilution.

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

Anti-NFIB / NF1B2 + NFIC/CTF antibody [EPR14504] (ab200829) at 1/10000 dilution + HepG2 (Human liver hepatocellular carcinoma cell line) at 20 µg

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

Predicted band size: 56, 47 kDa
Observed band size: 45-65 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking/Dilution buffer:5% NFDM /TBST

Based on the sequence analysis, ab200829 recognizes six isoforms within human with predicted Mw’s of 56KDa, 55KDa, 48KDa, 45KDa, 48KDa and 49KDa, respectively.

All lanes : Anti-NFIB / NF1B2 + NFIC/CTF antibody [EPR14504] (ab200829) at 1/2000 dilution

Lane 1 : Mouse heart lysate
Lane 2 : Mouse kidney lysate
Lane 3 : Mouse spleen lysate
Lane 4 : Rat brain lysate
Lane 5 : Rat heart 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), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 56, 47 kDa
Observed band size: 45-65 kDa why is the actual band size different from the predicted?


Exposure time: 1 minute

Blocking/Dilution buffer: 5% NFDM/TBST.

Based on the sequence analysis, ab200829 recognizes six isoforms within human with predicted Mw’s of 56KDa, 55KDa, 48KDa, 45KDa, 48KDa and 49KDa, respectively.