Recombinant Anti-NFkB p105 / p50 antibody [E381] (ab32360)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-These buffers may be stored at 4°C for several weeks or aliquoted and stored at -20°C for up to a year.	-Rabbit (1:1000) -To reduce and denature your samples, boil each cell lysate in sample buffer at 100°C for 5 min. Lysates can be aliquoted and stored at -20°C for future use.

Upstream tips
-These buffers may be stored at 4°C for several weeks or aliquoted and stored at -20°C for up to a year.
Protocol tips
-Rabbit (1:1000) -To reduce and denature your samples, boil each cell lysate in sample buffer at 100°C for 5 min. Lysates can be aliquoted and stored at -20°C for future use.

Publication protocol

SVGA cells and ReNcell CX cell-derived neurons were treated with 0-, 0.1-, 0.5-, 1-, 5-, and 10-ng/ml doses of human TNF-α (PeproTech) or human LIGHT (PeproTech). At 24 h posttreatment, cells were harvested and lysed on ice with 50 μl of in-house lysis buffer (0.05 M Tris [pH 7.4], 0.15 M NaCl, 0.002 M EDTA, 10% glycerol, and 1% NP-40 in ultrapure water) to extract proteins. The lysis buffer was supplemented with 1× HALT protease and phosphatase inhibitor cocktail (Thermo Scientific). A bicinchoninic acid (BCA) assay (Thermo Scientific) was used to determine the protein content of each sample per the manufacturer's instructions. Cell lysates were prepared for SDS-PAGE and heated at 95°C for 10 min. Proteins (15 μg per lane) were separated by SDS-PAGE on a 10% polyacrylamide gel and then transferred to a nitrocellulose membrane. The membrane was blocked in 5% skim milk solution for 1 h and then probed with the desired primary antibody (1:1,000 dilution) overnight at 4°C, followed by incubation at room temperature for 3 h. Primary antibodies used were as follows: mouse anti-human ERVK2 RT (H00002087-A01; Abnova), rabbit anti-human IRF1 (SC497; Santa Cruz), rabbit anti-human NF-κB p65 (ab7970; Abcam), rabbit anti-human NF-κB p50 (ab32360; Abcam), rabbit anti-human NF-κB p52 (4882S; Cell Signaling), and mouse anti-human β-actin (MA5-15739; Thermo Pierce) (loading control). The membrane was then probed with horseradish peroxidase-conjugated goat anti-mouse or -rabbit IgG secondary antibody (1:5,000 dilution) (170-6516 and 170-6515; Bio-Rad) for 2 h at room temperature. The membrane was developed with 2 ml of Luminata Crescendo Western HRP substrate (Millipore) and imaged using a Bio-Rad ChemiDoc XRS+ or Protein Simple FluorChem M chemiluminescence imager. Image Lab software was used to determine the molecular weight and relative density (normalized to that of β-actin) of each band.

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Manufacturer protocol

Download the product protocol from Abcam for Recombinant Anti-NFkB p105 / p50 antibody [E381] (ab32360) below.

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