Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: IKK gamma/NEMO knockout HAP1 cell lysate (20 µg)
Lane 3: Jurkat cell lysate (20 µg)
Lane 4: K562 cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab178872 observed at 40, 45, 50 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab178872 was shown to react with IKK gamma in wild-type HAP1 cells along with additional cross-reactive bands. No band was observed when IKK gamma/NEMO knockout samples were examined. Wild-type and IKK gamma/NEMO knockout samples were subjected to SDS-PAGE. ab178872 and ab8245 (loading control to GAPDH) were diluted at 1/5000 and 1/10,000 respectively and incubated overnight at 4°C. 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 1/10,000 dilution for 1 hour at room temperature before imaging.

Immunofluorescence analysis of 4% paraformaldehyde fixed, 0.1% Triton X-100 permeabilized NIH/3T3 cells (Mouse embyro fibroblast cells) labeling IKK gamma/NEMO (green) with ab178872 at 1/250 dilution showing cytoplasm and nucleus staining. Secondary ab: Goat anti rabbit IgG (Alexa Fluor® 488) (ab150077) at 1/200 dilution. Counter stain is labeling tubulin (red) with ab7291 at 1/500 dilution with secondary antibody Goat anti-Mouse AlexaFluor® 594 (ab150120) at 1/400 dilution. DAPI stains the nucleus in blue. -ve control 1 is ab178872 at 1/250 dilution, ab150120 at 1/400 dilution. -ve control 2 is ab7291 at 1/500 dilution, ab150077 at 1/200 dilution.

Immunohistochemical analysis of paraffin embedded human colonic adenocarcinoma tissue labeling IKK gamma/NEMO with ab178872 at 1/100 dilution followed by prediluted HRP Polymer for Rabbit/Mouse IgG. Cytoplasmic staining on colonic adenocarcinoma is observed.

Negative control: Using PBS instead of primary ab, secondary ab ImmunoHistoprobe (Ready to use) HRP Polymer for Rabbit/Mouse IgG.

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

IKK gamma/NEMO was immunoprecipitated from 1mg of HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell extract with ab178872 at 1/50 dilution. Western blot was performed of the immunoprecipitate using ab178872 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. Left lane: HeLa whole cell extract. Right lane:  PBS instead of HeLa whole cell extract.

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM/TBST.

All lanes : Anti-IKK gamma/NEMO antibody [EPR16629] (ab178872) at 1/1000 dilution

Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : IKBKG knockout HEK-293T cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 48 kDa
Observed band size: 48 kDa

Lanes 1- 2: Merged signal (red and green). Green - ab178872 observed at 48 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab178872 was shown to react with IKK gamma/NEMO in wild-type HEK-293T cells in western blot. The band observed in knockout cell line ab266674 (knockout cell lysate ab257153) lane below 48kDa may represent truncated forms and cleaved fragments. This has not been investigated further. Wild-type HEK-293T and IKBKG knockout HEK-293T cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab178872 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at a 1 in 1000 dilution and a 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-IKK gamma/NEMO antibody [EPR16629] (ab178872) at 1/5000 dilution

Lane 1 : Human fetal brain
Lane 2 : Human fetal kidney
Lane 3 : Human colon cancer

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 48 kDa
Additional bands at: 37,48,56 kDa (possible isoform)

Blocking buffer and concentration: 5% NFDM/TBST

Diluting buffer and concentration: 5% NFDM /TBST

 

ab178872 could recognize 3 isoforms with the predicted MWs of 37KDa, 56KDa and 48KDa, respectively.

All lanes : Anti-IKK gamma/NEMO antibody [EPR16629] (ab178872) at 1/20000 dilution

Lane 1 : HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysates
Lane 2 : K562 (Human chronic myelogenous leukemia cells from bone marrow) whole cell lysates
Lane 3 : Jurkat (Human T cell leukemia cells from peripheral blood) whole cell lysates

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: 48 kDa
Observed band size: 46-60 kDa why is the actual band size different from the predicted?

Blocking buffer and concentration: 5% NFDM/TBST

Diluting buffer and concentration: 5% NFDM /TBST

 

ab178872 could recognize 3 isoforms with the predicted MWs of 37KDa, 56KDa and 48KDa, respectively.

All lanes : Anti-IKK gamma/NEMO antibody [EPR16629] (ab178872) at 1/20000 dilution

Lane 1 : Mouse brain
Lane 2 : Mouse heart
Lane 3 : Mouse kidney
Lane 4 : Mouse spleen
Lane 5 : Rat brain
Lane 6 : Rat heart
Lane 7 : Rat kidney
Lane 8 : Rat spleen
Lane 9 : C6 (Rat glial tumor cells) whole cell lysates
Lane 10 : RAW 264.7 (Mouse macrophage cells transformed with Abelson murine leukemia virus) whole cell lysates
Lane 11 : PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysates
Lane 12 : NIH/3T3 (Mouse embyro fibroblast cells) whole cell lysates

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: 48 kDa
Observed band size: 37-60 kDa why is the actual band size different from the predicted?

Blocking buffer and concentration: 5% NFDM/TBST

Diluting buffer and concentration: 5% NFDM /TBST

Immunofluorescence analysis of 4% paraformaldehyde fixed, 0.1% Triton X-100 permeabilized HeLa cells (Human epithelial cells from cervix adenocarcinoma) labeling IKK gamma/NEMO (green) with ab178872 at 1/250 dilution showing cytoplasm and nucleus staining. Secondary ab: Goat anti rabbit IgG (Alexa Fluor® 488) (ab150077) at 1/200 dilution. Counter stain is labeling tubulin (red) with ab7291 at 1/500 dilution with secondary antibody Goat anti-Mouse AlexaFluor® 594 (ab150120) at 1/400 dilution. DAPI stains the nucleus in blue. -ve control 1 is ab178872 at 1/250 dilution, ab150120 at 1/400 dilution. -ve control 2 is ab7291 at 1/500 dilution, ab150077 at 1/200 dilution.

 

Immunohistochemical analysis of paraffin embedded Rat colon tissue labeling IKK gamma/NEMO with ab178872 at 1/100 dilution followed by prediluted HRP Polymer for Rabbit/Mouse IgG. Counter stain is Hematoxylin. Cytoplasm staining on epithelial cells of rat colon is observed.

Negative control: Using PBS instead of primary ab, secondary ab as above.

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