Recombinant Anti-IKB alpha antibody [E130] (ab32518)

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

Lanes 1 - 3: Merged signal (red and green). Green - ab32518 observed at 38 kDa. Red - loading control, ab18058, observed at 130 kDa.

ab32518 was shown to specifically react with IKB alpha in wild-type HAP1 cells. No band was observed when IKB alpha knockout samples were tested. Wild-type and IKB alpha knockout samples were subjected to SDS-PAGE. ab32518 and ab18058 (Mouse anti Vinculin loading control) were incubated overnight at 4°C at 1/10,000 dilution and 1/20,000 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 1/20,000 dilution for 1 hour at room temperature before imaging.

Immunohistochemical staining of paraffin embedded human stomach with purified ab32518 at a working dilution of 1 in 100.

The secondary antibody used is a HRP polymer for rabbit IgG. The sample is counterstained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0.

PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

Unpurified ab32518 staining IKB alpha in RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) cells treated with FK506 (ab120223), by ICC/IF. Decrease in IkBα/&beta expression correlates with increased concentration of FK506, as described in literature.
The cells were incubated at 37°C for 3h in media containing different concentrations of ab120223 (FK506) in DMSO, fixed with 100% methanol for 5 minutes at -20°C and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature.

Staining of the treated cells with ab32518 (1/100 dilution) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight^® 488 goat anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

Immunofluorescence staining of HeLa (Human epithelial cell line from cervix adenocarcinoma) cells with purified ab32518 at a working dilution of 1 in 50, counterstained with DAPI.

The secondary antibody was ab150077, Alexa Fluor^® 488 goat anti rabbit, used at a dilution of 1 in 500. The cells were fixed in 4% PFA and permeabilized using 0.1% Triton X 100.

The negative control is shown in bottom right hand panel - for the negative control, purified ab32518 was used at a dilution of 1/50 followed by ab150120, Alexa Fluor^® 594 goat anti-mouse antibody at a dilution of 1/500.

ab32518 (purified) at 1/20 immunoprecipitating IKB alpha in HeLa (Human epithelial cell line from cervix adenocarcinoma) cell lysate (Lane 1). For western blotting a HRP-conjugated goat anti-rabbit IgG was used as the secondary antibody (1/1000).

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

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunohistochemical analysis of paraffin-embedded human prostate carcinoma using unpurified ab32518 at 1/50 dilution.

Intracellular Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling IKB alpha with purified ab32518 at 1/20 dilution (10µg/mL) (red). 

Cells were fixed with 4% paraformaldehyde and permeabilized with 90% methanol. A Goat anti rabbit IgG (Alexa Fluorr^® 488) (1/2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) were used as the unlabeled control. 

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunohistochemical staining of paraffin embedded rat kidney with purified ab32518 at a working dilution of 1 in 100.

The secondary antibody used is a HRP polymer for rabbit IgG. The sample is counterstained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0.

PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

ICC/IF image of unpurified ab32518 stained HeLa (Human epithelial cell line from cervix adenocarcinoma) cells.

The cells were fixed in 100% methanol (5 min) and then incubated in 1% BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilize the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab32518, 1/1000 dilution) overnight at +4°C. The secondary antibody (green) was Alexa Fluor^® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor^® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h.

DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43 µM.

Blocking/Dilution buffer: 5% NFDM/TBST.

Blocking buffer: 5% NFDM/TBST

Dilution buffer: 5% NFDM/TBST

Unpurified ab32518 used to immunoprecipitate IKB alpha from HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate. The antibody was further used to Western blot the protein.

Lane 1: IKB alpha IP

Lane 2: Control immunoprecipitate

Lane 3: Input (20%)

See Abreview

Unpurified ab32518 used to immunoprecipitate IKB alpha from PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate. The antibody was further used to Western blot the protein.

Lane 1: IKB alpha IP

Lane 2: Control immunoprecipitate

Lane 3: Input (20%)

See Abreview

Unpurified ab32518 at 1/100 staining mouse kidney tissue sections by IHC-P.

The tissue was paraformaldehyde fixed and a heat mediated antigen retrieval step in citrate buffer was performed before the tissue was blocked and incubated with the antibody for 1 hour. An HRP conjugated goat anti-rabbit antibody was used as the secondary.

See Abreview