Anti-IKB beta antibody [EPR5037] (ab109509) at 1/1000 dilution (Purified) + HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates at 15 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 37 kDa
Observed band size: 48 kDa why is the actual band size different from the predicted?

Lane 1: Wild type HAP1 whole cell lysate (20 µg)
Lane 2: IKB beta knockout HAP1 whole cell lysate (20 µg)
Lane 3: HeLa whole cell lysate (20 µg)

Lanes 1 - 3: Merged signal (red and green). Green - unpurified ab109509 observed at 45 kDa. Red - loading control, ab18058, observed at 130 kDa.

ab109509 was shown to specifically react with IKB beta in wild type cells as signal was lost in IKB beta knockout cells. Wild-type and IKB beta knockout samples were subjected to SDS-PAGE. ab109509 and ab18058 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

ab109509 (purified) at 1:20 dilution (1 µg) immunoprecipitating IKB beta in HeLa whole cell lysate.

Lane 1 (input): HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10ug
Lane 2 (+): ab109509 & HeLa whole cell lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab109509 in HeLa whole cell lysate

For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1:1000 dilution.

Blocking and diluting buffer: 5% NFDM/TBST.

Flow Cytometry analysis of Jurkat (Human T cell leukemia T lymphocyte) cells labeling IKB beta with purified ab109509 at 1:20 dilution (10 µg/ml) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) secondary antibody was used at 1:2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

Immunocytochemistry/ Immunofluorescence analysis of Jurkat (Human T cell leukemia T lymphocyte) cells labeling IKB beta with purified ab109509 at 1:50 dilution (2.0 µg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1:200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1:1000 (2 µg/ml) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human kidney tissue sections labeling IKB beta with purified ab109509 at 1:500 dilution (0.20 µg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

Immunohistochemical analysis of paraffin-embedded Human kidney tissue using unpurified ab109509 at a dilution of 1/100.

Heat mediated antigen retrieval was performed with citrate buffer pH 6 before commencing with IHC staining protocol.

All lanes : Anti-IKB beta antibody [EPR5037] (ab109509) at 1/1000 dilution (Unpurified)

Lane 1 : Jurkat cell lysate
Lane 2 : HeLa cell lysate
Lane 3 : THP-1 cell lysate
Lane 4 : MCF7 cell lysate

Lysates/proteins at 10 µg per lane.

Predicted band size: 37 kDa
Observed band size: 48 kDa why is the actual band size different from the predicted?