Anti-NFkB p100/NFKB2 antibody [EPR4686-66] - BSA and Azide free

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-NFkB p100/NFKB2 antibody [EPR4686-66] - BSA and Azide free (AB249847)

This data was developed using ab175192, the same antibody clone in a different buffer formulation.

ab175192 staining NFkB p100/NFKB2 in wild-type HAP1 cells (top panel) and NFkB p100/NFKB2 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 ab175192 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.

• IP

Supplier Data

Immunoprecipitation - Anti-NFkB p100/NFKB2 antibody [EPR4686-66] - BSA and Azide free (AB249847)

This data was developed using ab175192, the same antibody clone in a different buffer formulation.

Western blot analysis on immunoprecipitation pellet from Jurkat cell lysate labeling NFkB p100/NFKB2 with ab175192 at 1/10 dilution.

All lanes:

Immunoprecipitation - Anti-NFkB p100/NFKB2 antibody [EPR4686-66] (<a href='/en-us/products/primary-antibodies/nfkb-p100-nfkb2-antibody-epr4686-66-ab175192'>ab175192</a>)

Predicted band size: 97 kDa

false

• WB

Supplier Data

Western blot - Anti-NFkB p100/NFKB2 antibody [EPR4686-66] - BSA and Azide free (AB249847)

This data was developed using ab175192, the same antibody clone in a different buffer formulation.

All lanes:

Western blot - Anti-NFkB p100/NFKB2 antibody [EPR4686-66] (<a href='/en-us/products/primary-antibodies/nfkb-p100-nfkb2-antibody-epr4686-66-ab175192'>ab175192</a>) at 1/10000 dilution

Lane 1:

Jurkat cell lysate at 10 µg

Lane 2:

HeLa cell lysate at 10 µg

Lane 3:

Daudi cell lysates at 10 µg

Lane 4:

MCF7 cell lysate at 10 µg

Predicted band size: 97 kDa

false

• WB

Lab

Western blot - Anti-NFkB p100/NFKB2 antibody [EPR4686-66] - BSA and Azide free (AB249847)

This data was developed using the same antibody clone in a different buffer formulation (ab175192).

Lanes 1- 2 : Merged signal (red and green). Green - ab175192 observed at 120 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

ab175192 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 HCT117 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. ab175192 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at a 1 in 10000 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 [EPR4686-66] (<a href='/en-us/products/primary-antibodies/nfkb-p100-nfkb2-antibody-epr4686-66-ab175192'>ab175192</a>) at 1/1000 dilution

Lane 1:

Wild-type 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>)

Lane 2:

NFKB2 CRISPR/Cas9 edited HCT116 cell lysate at 20 µg

Predicted band size: 97 kDa

Observed band size: 120 kDa

false

• WB

Lab

Western blot - Anti-NFkB p100/NFKB2 antibody [EPR4686-66] - BSA and Azide free (AB249847)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab175192).

Lanes 1- 2 : Merged signal (red and green). Green - ab175192 observed at 120 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

ab175192 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. ab175192 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at a 1 in 10000 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 [EPR4686-66] (<a href='/en-us/products/primary-antibodies/nfkb-p100-nfkb2-antibody-epr4686-66-ab175192'>ab175192</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 line (<a href='/en-us/products/cell-lines/human-nfkb2-nfkb-p100-nfkb2-knockout-hep-g2-cell-line-ab262323'>ab262323</a>)

Lane 2:

NFKB2 CRISPR/Cas9 edited HepG2 cell lysate at 20 µg

Predicted band size: 97 kDa

Observed band size: 120 kDa

false

• WB

Lab

Western blot - Anti-NFkB p100/NFKB2 antibody [EPR4686-66] - BSA and Azide free (AB249847)

Lanes 1- 2 : Merged signal (red and green). Green - ab175192 observed at 120 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

ab175192 was shown to react with NFkB p100/NFKB2 in wild-type HCT116 cells in western blot. The band observed in knockout cell line ab266883 (knockout cell lysate ab257245) lane below 97kDa may represent truncated forms and cleaved fragments. This has not been investigated further. Wild-type HCT116 and NFKB2 knockout HCT117 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. ab175192 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at a 1 in 10000 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 [EPR4686-66] (<a href='/en-us/products/primary-antibodies/nfkb-p100-nfkb2-antibody-epr4686-66-ab175192'>ab175192</a>) at 1/1000 dilution

Lane 1:

Wild-type HCT116 cell lysate at 20 µg

Lane 2:

Western blot - Human NFKB2 (NFkB p100/NFKB2) knockout HCT116 cell lysate (<a href='/en-us/products/cell-lysates/human-nfkb2-nfkb-p100-nfkb2-knockout-hct116-cell-lysate-ab257245'>ab257245</a>)

Predicted band size: 97 kDa

Observed band size: 120 kDa

false

• WB

Lab

Western blot - Anti-NFkB p100/NFKB2 antibody [EPR4686-66] - BSA and Azide free (AB249847)

This data was developed using ab175192, 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 - ab175192 observed at 97 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab175192 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. ab175192 and ab8245 (loading control to GAPDH) were diluted 1/10 000 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 [EPR4686-66] (<a href='/en-us/products/primary-antibodies/nfkb-p100-nfkb2-antibody-epr4686-66-ab175192'>ab175192</a>)

Predicted band size: 97 kDa

false