Anti-NFkB p100/NFKB2 antibody [EPR4686] - BSA and Azide free

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-NFkB p100/NFKB2 antibody [EPR4686] - BSA and Azide free (AB174482)

This data was developed using ab109440, the same antibody clone in a different buffer formulation.

ab109440 staining NFkB p100/p52 in wild-type HAP1 cells (top panel) and NFkB p100/p52 knockout HAP1 cells (bottom panel). The cells were fixed with 4% formaldehyde (10min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab109440 at 1/250 dilution and ab195889 at 1/250 dilution (shown in pseudo colour red) overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

• WB

Lab

Western blot - Anti-NFkB p100/NFKB2 antibody [EPR4686] - BSA and Azide free (AB174482)

This data was developed using ab109440, the same antibody clone in a different buffer formulation.

Lane 1 : Wild-type HAP1 cell lysate (20 μg)

Lane 2 : NFκB p100 knockout HAP1 cell lysate (20 μg)

Lane 3 : Jurkat cell lysate (20 μg)

Lane 4 : HeLa cell lysate (20 μg)

Lanes 1 - 4 : Merged signal (red and green). Green - ab109440 observed at 100 kDa. Red - loading control, ab8245, observed at 37 kDa.
ab109440 was shown to specifically react with NFκB p100 when NFκB p100 knockout samples were used. Wild-type and NFκB p100 knockout samples were subjected to SDS-PAGE. ab109440 and ab109440 (loading control to GAPDH) were diluted 1/10000 and 1/2000 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 1/10000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-NFkB p100/NFKB2 antibody [EPR4686] (<a href='/en-us/products/primary-antibodies/nfkb-p100-nfkb2-antibody-epr4686-ab109440'>ab109440</a>)

Predicted band size: 97 kDa

false

• WB

Lab

Western blot - Anti-NFkB p100/NFKB2 antibody [EPR4686] - BSA and Azide free (AB174482)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab109440).

Lanes 1- 2 : Merged signal (red and green). Green - ab109440 observed at 120 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

ab109440 was shown to react with NFkB p100/NFKB2 in wild-type HepG2 cells in western blot. The band observed in CRISPR/Cas9 edited cell line ab262323 (CRISPR/Cas9 edited cell lysate ab257247) lane below 97kDa may represent truncated forms and cleaved fragments. This has not been investigated further. Wild-type HepG2 and NFKB2 CRISPR/Cas9 edited HepG2 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. ab109440 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-NFkB p100/NFKB2 antibody [EPR4686] (<a href='/en-us/products/primary-antibodies/nfkb-p100-nfkb2-antibody-epr4686-ab109440'>ab109440</a>) at 1/1000 dilution

Lane 1:

Wild-type HepG2 cell lysate at 20 µg

Lane 2:

Western blot - Human NFKB2 (NFkB p100/NFKB2) knockout Hep G2 cell lysate (<a href='/en-us/products/cell-lysates/human-nfkb2-nfkb-p100-nfkb2-knockout-hep-g2-cell-lysate-ab257247'>ab257247</a>) at 20 µg

Secondary

Lanes 1 - 2:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/20000 dilution

Lanes 1 - 2:

Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/20000 dilution

Predicted band size: 30 kDa,97 kDa

Observed band size: 120 kDa,30 kDa,37 kDa

false

• WB

Lab

Western blot - Anti-NFkB p100/NFKB2 antibody [EPR4686] - BSA and Azide free (AB174482)

This data was developed using the same antibody clone in a different buffer formulation (ab109440).

Lanes 1- 2 : Merged signal (red and green). Green - ab109440 observed at 120 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

ab109440 was shown to react with NFkB p100/NFKB2 in wild-type HCT116 cells in western blot. The band observed in CRISPR/Cas9 edited cell line ab266883 (CRISPR/Cas9 edited cell lysate ab257245) lane below 97kDa may represent truncated forms and cleaved fragments. This has not been investigated further. Wild-type HCT116 and NFKB2 CRISPR/Cas9 edited HCT116 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. ab109440 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-NFkB p100/NFKB2 antibody [EPR4686] (<a href='/en-us/products/primary-antibodies/nfkb-p100-nfkb2-antibody-epr4686-ab109440'>ab109440</a>) at 1/1000 dilution

Lane 1:

Wild-type HCT116 cell lysate at 20 µg

Lane 2:

NFKB2 CRISPR/Cas9 edited HCT116 cell lysate at 20 µg

Lane 2:

Western blot - Human NFKB2 (NFkB p100/NFKB2) knockout HCT116 cell line (<a href='/en-us/products/cell-lines/human-nfkb2-nfkb-p100-nfkb2-knockout-hct116-cell-line-ab266883'>ab266883</a>)

Predicted band size: 97 kDa

Observed band size: 120 kDa

false

• WB

Unknown

Western blot - Anti-NFkB p100/NFKB2 antibody [EPR4686] - BSA and Azide free (AB174482)

This data was developed using ab109440, the same antibody clone in a different buffer formulation.

All lanes:

Western blot - Anti-NFkB p100/NFKB2 antibody [EPR4686] (<a href='/en-us/products/primary-antibodies/nfkb-p100-nfkb2-antibody-epr4686-ab109440'>ab109440</a>) at 1/10000 dilution

Lane 1:

Jurkat cell lysate at 10 µg

Lane 2:

HeLa cell lysate at 10 µg

Lane 3:

ECV-304 cell lysate at 10 µg

Lane 4:

MCF7 cell lysate at 10 µg

Predicted band size: 97 kDa

Observed band size: 110 kDa

false