Anti-IKK gamma/NEMO antibody [EPR16629]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-IKK gamma/NEMO antibody [EPR16629] (AB178872)

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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IKK gamma/NEMO antibody [EPR16629] (AB178872)

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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IKK gamma/NEMO antibody [EPR16629] (AB178872)

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.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-IKK gamma/NEMO antibody [EPR16629] (AB178872)

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.

• WB

Lab

Western blot - Anti-IKK gamma/NEMO antibody [EPR16629] (AB178872)

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:

Western blot - Anti-IKK gamma/NEMO antibody [EPR16629] (ab178872) at 1/1000 dilution

Lane 1:

Wild-type HEK-293T cell lysate at 20 µg

Lane 2:

IKBKG knockout HEK-293T cell lysate at 20 µg

Predicted band size: 48 kDa

Observed band size: 48 kDa

false

• WB

Supplier Data

Western blot - Anti-IKK gamma/NEMO antibody [EPR16629] (AB178872)

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:

Western blot - Anti-IKK gamma/NEMO antibody [EPR16629] (ab178872) at 1/20000 dilution

Lane 1:

HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysates at 20 µg

Lane 2:

K562 (Human chronic myelogenous leukemia cells from bone marrow) whole cell lysates at 20 µg

Lane 3:

Jurkat (Human T cell leukemia cells from peripheral blood) whole cell lysates at 20 µg

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

false

• WB

Supplier Data

Western blot - Anti-IKK gamma/NEMO antibody [EPR16629] (AB178872)

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:

Western blot - Anti-IKK gamma/NEMO antibody [EPR16629] (ab178872) at 1/5000 dilution

Lane 1:

Human fetal brain at 10 µg

Lane 2:

Human fetal kidney at 10 µg

Lane 3:

Human colon cancer at 10 µg

Secondary

All lanes:

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

Predicted band size: 48 kDa

Observed band size: 37 kDa,48 kDa,56 kDa

false

• IP

Supplier Data

Immunoprecipitation - Anti-IKK gamma/NEMO antibody [EPR16629] (AB178872)

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:

Immunoprecipitation - Anti-IKK gamma/NEMO antibody [EPR16629] (ab178872)

Predicted band size: 48 kDa

Observed band size: 37-60 kDa

false

• WB

Lab

Western blot - Anti-IKK gamma/NEMO antibody [EPR16629] (AB178872)

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.

All lanes:

Western blot - Anti-IKK gamma/NEMO antibody [EPR16629] (ab178872)

Predicted band size: 48 kDa

false

• WB

Lab

Western blot - Anti-IKK gamma/NEMO antibody [EPR16629] (AB178872)

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 CRISPR/Cas9 edited cell line ab266674 (CRISPR/Cas9 edited 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 CRISPR/Cas9 edited 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:

Western blot - Anti-IKK gamma/NEMO antibody [EPR16629] (ab178872) at 1/1000 dilution

Lane 1:

Wild-type HEK-293T cell lysate at 20 µg

Lane 2:

IKBKG CRISPR/Cas9 edited HEK-293T cell lysate at 20 µg

Lane 2:

Western blot - Human IKBKG (IKK gamma/NEMO) knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-ikbkg-ikk-gamma-nemo-knockout-hek-293t-cell-line-ab266674'>ab266674</a>)

Predicted band size: 48 kDa

Observed band size: 48 kDa

false

• WB

Unknown

Western blot - Anti-IKK gamma/NEMO antibody [EPR16629] (AB178872)

Blocking buffer and concentration : 5% NFDM/TBST

Diluting buffer and concentration : 5% NFDM /TBST

All lanes:

Western blot - Anti-IKK gamma/NEMO antibody [EPR16629] (ab178872) at 1/20000 dilution

Lane 1:

Mouse brain at 10 µg

Lane 2:

Mouse heart at 10 µg

Lane 3:

Mouse kidney at 10 µg

Lane 4:

Mouse spleen at 10 µg

Lane 5:

Rat brain at 10 µg

Lane 6:

Rat heart at 10 µg

Lane 7:

Rat kidney at 10 µg

Lane 8:

Rat spleen at 10 µg

Lane 9:

C6 (Rat glial tumor cells) whole cell lysates at 10 µg

Lane 10:

RAW 264.7 (Mouse macrophage cells transformed with Abelson murine leukemia virus) whole cell lysates at 10 µg

Lane 11:

PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysates at 10 µg

Lane 12:

NIH/3T3 (Mouse embyro fibroblast cells) whole cell lysates at 10 µg

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

false