Anti-IKK gamma/NEMO antibody [EPR16629] - BSA and Azide free

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-IKK gamma/NEMO antibody [EPR16629] - BSA and Azide free (AB230832)

This ICC data was generated using the same anti-IKK gamma/NEMO antibody clone, EPR16629, in a different buffer formulation (cat# ab178872).

Immunofluorescence analysis of 4% paraformaldehyde fixed, 0.1% Triton X-100 permeabilized HeLa cells (Human epithelial cells from cervix adenocarcinoma) labeling IKK gamma (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] - BSA and Azide free (AB230832)

This IHC data was generated using the same anti-IKK gamma/NEMO antibody clone, EPR16629, in a different buffer formulation (cat# ab178872).

Immunohistochemical analysis of paraffin embedded human colonic adenocarcinoma tissue labeling IKK gamma 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.

Heat mediated antigen retrieval was performed 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] - BSA and Azide free (AB230832)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab178872).

Heat mediated antigen retrieval was performed 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] - BSA and Azide free (AB230832)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab178872).

• IP

Supplier Data

Immunoprecipitation - Anti-IKK gamma/NEMO antibody [EPR16629] - BSA and Azide free (AB230832)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab178872).

All lanes:

Immunoprecipitation - Anti-IKK gamma/NEMO antibody [EPR16629] (<a href='/en-us/products/primary-antibodies/ikk-gamma-nemo-antibody-epr16629-ab178872'>ab178872</a>)

Predicted band size: 48 kDa

Observed band size: 37-60 kDa

false

• WB

Lab

Western blot - Anti-IKK gamma/NEMO antibody [EPR16629] - BSA and Azide free (AB230832)

This WB data was generated using the same anti-IKK gamma antibody clone, EPR16629, in a different buffer formulation (cat# 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] (<a href='/en-us/products/primary-antibodies/ikk-gamma-nemo-antibody-epr16629-ab178872'>ab178872</a>)

Predicted band size: 48 kDa

false

• WB

Lab

Western blot - Anti-IKK gamma/NEMO antibody [EPR16629] - BSA and Azide free (AB230832)

This data was developed using the same antibody clone in a different buffer formulation (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] (<a href='/en-us/products/primary-antibodies/ikk-gamma-nemo-antibody-epr16629-ab178872'>ab178872</a>) 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