Human TNF knockout THP-1 cell line (ab273761)

This Western blot image is a comparison between ab215188 and ab183218 tested under the same conditions. While ab215188 is suitable for WB for some samples, ab183218 was found to be more sensitive. False colour image of Western blot: Anti-TNF alpha antibody [EPR20972] staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab215188 was shown to bind specifically to TNF alpha. A band was observed at 27 kDa in treated U937 cell lysates with no signal observed at this size without treatment. No signal was observed in wild-type THP-1 cell lysates or in TNF knockout cell line ab273761 (knockout cell lysate ab275507) with ab215188. However, a band was observed at 27 kDa in treated wild-type THP-1 cell lysates with ab183218. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

Lanes 1 - 6: Merged signal (red and green). Green - ab255275 observed at 26 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55 kDa.

ab255275 was shown to react with TNF alpha in THP-1 wild-type cells in Western blot with loss of signal observed in TNF knockout sample. Wild-type and TNF knockout THP-1 cell lysates were subjected to SDS-PAGE. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with ab255275 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4 °C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.

Lanes 1 - 6: Merged signal (red and green). Green - ab183218 observed at 26 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55 kDa.

ab183218 was shown to react with TNF alpha in THP-1 wild-type cells in Western blot with loss of signal observed in TNF knockout sample. Wild-type and TNF knockout THP-1 cell lysates were subjected to SDS-PAGE. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with ab183218 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4 °C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.

Human TNF alpha concentration was interpolated from the standard curve. Supernatants from cell culture samples were serially diluted and assessed by the Human TNF alpha ELISA kit (ab181421). Wild-type THP-1 and TNF alpha knockout THP-1 (ab273761) cells were assessed in duplicate (n=2). Cells were either treated with 100 ng/ml LPS for 16 h to induce expression of TNF alpha or not treated with LPS. Data are represented as the mean and error bars represent standard deviation.”

Homozygous: 52 bp deletion in exon 4