NF-kB Signaling Pathway Antibody Sampler Panel

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - NF-kB Signaling Pathway Antibody Sampler Panel (AB228529)

ICC/IF image of unpurified ab32518 stained HeLa cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab32518, 1/1000 dilution) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor^® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43μM.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - NF-kB Signaling Pathway Antibody Sampler Panel (AB228529)

Immunohistochemical analysis of colon sections from mice, staining NF-kB p65 with ab32536.

Antigen retrieval was performed by microwave heating in citrate buffer, pH 6. Sections were incubated overnight with primary antibody (1/250) and staining was detected using ab80437 EXPOSE Rabbit specific HRP/DAB detection IHC kit.

• WB

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Western blot - NF-kB Signaling Pathway Antibody Sampler Panel (AB228529)

Predicted band : 105 kDa

Lane 1 : Wild-type HAP1 cell lysate (20 μg)
Lane 2 : NFkB p105 / p50 knockout HAP1 cell lysate (20 μg)
Lane 3 : HeLa cell lysate (20 μg)
Lane 4 : Jurkat cell lysate (20 μg)

Lanes 1 - 4 : Merged signal (red and green). Green - Anti-NFkB p105 / p50 antibody [E381] (ab32360) observed at 120, 50 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab32360 was shown to specifically react with NFkB p105 / p50 when NFkB p105 / p50 knockout samples were used. Wild-type and NFkB p105 / p50 knockout samples were subjected to SDS-PAGE. ab32360 and ab8245 (loading control to GAPDH) were diluted at 1/1000 and 1/10 000 respectively and 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 hour at room temperature before imaging.

All lanes:

Western blot - Anti-NFkB p105 / p50 antibody [E381] (<a href='/en-us/products/primary-antibodies/nfkb-p105-p50-antibody-e381-ab32360'>ab32360</a>)

Lane 1:

Wild-type HAP1 cell lysate at 20 µg

Lane 2:

NFkB p105 / p50 knockout HAP1 cell lysate at 20 µg

Lane 3:

HeLa cell lysate at 20 µg

Lane 4:

Jurkat cell lysate at 20 µg

Predicted band size: 105 kDa

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• WB

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Western blot - NF-kB Signaling Pathway Antibody Sampler Panel (AB228529)

Primary - Anti-IKB alpha (phospho S36) antibody [EPR6235(2)] (ab133462) at 1/10000 dilution

Lane 1 : HeLa cell lysate, 10 μg

Lane 2 : HeLa cell lysate treated with FBS and Calyculin A, 10 μg

Secondary - HRP labeled goat anti-rabbit IgG, 1/2000 dilution

Predicted band : 35 kDa

Actual band : 40 kDa

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• Dot

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Dot Blot - NF-kB Signaling Pathway Antibody Sampler Panel (AB228529)