Human TNF knockout THP-1 cell lysate

• WB

Lab

Western blot - Human TNF knockout THP-1 cell lysate (AB275507)

Lane 1 : Wild-type THP-1 Brefeldin A (ab120299) treated (5 ug/ml, 4 h) cell lysate 30 ug
Lane 2 : Wild-type THP-1 LPS treated (100 ng/ml, 16 h) and Brefeldin A (ab120299) treated (5 ug/ml, 4 h) cell lysate 30 ug
Lane 3 : TNF alpha knockout THP-1 Brefeldin A (ab120299) treated (5 ug/ml, 4 h) cell lysate 30 ug
Lane 4 : TNF alpha knockout THP-1 LPS treated (100 ng/ml, 16 h) and Brefeldin A (ab120299) treated (5 ug/ml, 4 h) cell lysate 30 ug
Lane 5 : U937 PMA treated (10 mM, 2 days) plus 16 h no treatment and Brefeldin A (ab120299) treated (5 ug/ml, 4 h) cell lysate 30 ug
Lane 6 : U937 PMA treated (10 mM, 2 days) and LPS treated (1 ug/ml, 16 h) plus Brefeldin A (ab120299) treated (5 ug/ml, 4 h) cell lysate 30 ug
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 55kDa.
ab255275 was shown to react with TNF alpha in wild-type THP-1 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.

All lanes:

Western blot - Anti-TNF alpha antibody [EPR22598-212] (<a href='/en-us/products/primary-antibodies/tnf-alpha-antibody-epr22598-212-ab255275'>ab255275</a>) at 1/1000 dilution

Lane 1:

Wild-type THP-1 Brefeldin A (<a href='/en-us/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>) treated (5 µg/ml, 4 h) cell lysate at 30 µg

Lane 2:

Wild-type THP-1 LPS treated (100 ng/ml, 16 h) and Brefeldin A (<a href='/en-us/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>) treated (5 µg/ml, 4 h) cell lysate at 30 µg

Lane 2:

Western blot - Human TNF knockout THP-1 cell line (<a href='/en-us/products/cell-lines/human-tnf-knockout-thp-1-cell-line-ab273761'>ab273761</a>)

Lane 3:

TNF alpha knockout THP-1 Brefeldin A (<a href='/en-us/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>) treated (5 µg/ml, 4 h) cell lysate at 30 µg

Lane 4:

TNF alpha knockout THP-1 LPS treated (100 ng/ml, 16 h) and Brefeldin A (<a href='/en-us/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>) treated (5 µg/ml, 4 h) cell lysate at 30 µg

Lane 5:

U937 PMA treated (10 mM, 2 days) plus 16 h no treatment and Brefeldin A (<a href='/en-us/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>) treated (5 µg/ml, 4 h) cell lysate at 30 µg

Lane 6:

U937 PMA treated (10 mM, 2 days) and LPS treated (1 µg/ml, 16 h) plus Brefeldin A (<a href='/en-us/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>) treated (5 µg/ml, 4 h) cell lysate at 30 µg

Predicted band size: 25 kDa

Observed band size: 26 kDa

false

• WB

Lab

Western blot - Human TNF knockout THP-1 cell lysate (AB275507)

Lane 1 : Wild-type THP-1 control : Brefeldin A (5 μg/mL, 4 h) cell lysate 30 μg
Lane 2 : Wild-type treated THP-1 : LPS (100 ng/mL, 16 h), Brefeldin A (5 μg/mL, last 4 h) cell lysate 30 μg
Lane 3 : TNF alpha knockout THP-1 control : Brefeldin A (5 μg/mL, 4 h) cell lysate 30 μg
Lane 4 : TNF alpha knockout THP-1 treated : LPS (100 ng/mL, 16 h), Brefeldin A (5 μg/mL, last 4 h) cell lysate 30 μg
Lane 5 : U937 control : PMA (10 mM, 2 days), Brefeldin A (5 μg/mL, last 4 h) cell lysate 30 μg
Lane 6 : U937 treated : PMA (10 mM, 2 days), LPS (1 μg/mL, last 16 h), Brefeldin A (5 μg/mL, last 4 h) cell lysate 30 μg
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.

All lanes:

Western blot - Anti-TNF alpha antibody [EPR20972] (<a href='/en-us/products/primary-antibodies/tnf-alpha-antibody-epr20972-ab215188'>ab215188</a>) at 1/1000 dilution

Lane 1:

Wild-type THP-1 control: Brefeldin A (5 ug/mL, 4 h) cell lysate at 30 µg

Lane 2:

Wild-type treated THP-1: LPS (100 ng/mL, 16 h), Brefeldin A (5 ug/mL, last 4 h) cell lysate at 30 µg

Lane 2:

Western blot - Human TNF knockout THP-1 cell line (<a href='/en-us/products/cell-lines/human-tnf-knockout-thp-1-cell-line-ab273761'>ab273761</a>)

Lane 3:

TNF alpha knockout THP-1 control: Brefeldin A (5 ug/mL, 4 h) cell lysate at 30 µg

Lane 4:

TNF alpha knockout THP-1 treated: LPS (100 ng/mL, 16 h), Brefeldin A (5 ug/mL, last 4 h) cell lysate at 30 µg

Lane 5:

U937 control: PMA (10 mM, 2 days), Brefeldin A (5 ug/mL, last 4 h) cell lysate at 30 µg

Lane 6:

U937 treated: PMA (10 mM, 2 days), LPS (1 ug/mL, last 16 h), Brefeldin A (5 ug/mL, last 4 h) cell lysate at 30 µg

Predicted band size: 25 kDa

Observed band size: 27 kDa

false

• WB

Lab

Western blot - Human TNF knockout THP-1 cell lysate (AB275507)

Lane 1 : Wild-type THP-1 Brefeldin A (ab120299) treated (5 ug/ml, 4 h) cell lysate 30 ug
Lane 2 : Wild-type THP-1 LPS treated (100 ng/ml, 16 h) and Brefeldin A (ab120299) treated (5 ug/ml, 4 h) cell lysate 30 ug
Lane 3 : TNF alpha knockout THP-1 Brefeldin A (ab120299) treated (5 ug/ml, 4 h) cell lysate 30 ug
Lane 4 : TNF alpha knockout THP-1 LPS treated (100 ng/ml, 16 h) and Brefeldin A (ab120299) treated (5 ug/ml, 4 h) cell lysate 30 ug
Lane 5 : U937 PMA treated (10 mM, 2 days) plus 16 h no treatment and Brefeldin A (ab120299) treated (5 ug/ml, 4 h) cell lysate 30 ug
Lane 6 : U937 PMA treated (10 mM, 2 days) and LPS treated (1 ug/ml, 16 h) plus Brefeldin A (ab120299) treated (5 ug/ml, 4 h) cell lysate 30 ug
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 55kDa.
ab183218 was shown to react with TNF alpha in wild-type THP-1 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.

All lanes:

Western blot - Anti-TNF alpha antibody [EPR19147] (<a href='/en-us/products/primary-antibodies/tnf-alpha-antibody-epr19147-ab183218'>ab183218</a>) at 1/1000 dilution

Lane 1:

Wild-type THP-1 Brefeldin A (<a href='/en-us/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>) treated (5 µg/ml, 4 h) cell lysate at 30 µg

Lane 2:

Wild-type THP-1 LPS treated (100 ng/ml, 16 h) and Brefeldin A (<a href='/en-us/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>) treated (5 µg/ml, 4 h) cell lysate at 30 µg

Lane 2:

Western blot - Human TNF knockout THP-1 cell line (<a href='/en-us/products/cell-lines/human-tnf-knockout-thp-1-cell-line-ab273761'>ab273761</a>)

Lane 3:

TNF alpha knockout THP-1 Brefeldin A (<a href='/en-us/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>) treated (5 µg/ml, 4 h) cell lysate at 30 µg

Lane 4:

TNF alpha knockout THP-1 LPS treated (100 ng/ml, 16 h) and Brefeldin A (<a href='/en-us/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>) treated (5 µg/ml, 4 h) cell lysate at 30 µg

Lane 5:

U937 PMA treated (10 mM, 2 days) plus 16 h no treatment and Brefeldin A (<a href='/en-us/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>) treated (5 µg/ml, 4 h) cell lysate at 30 µg

Lane 6:

U937 PMA treated (10 mM, 2 days) and LPS treated (1 µg/ml, 16 h) plus Brefeldin A (<a href='/en-us/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>) treated (5 µg/ml, 4 h) cell lysate at 30 µg

Predicted band size: 25 kDa

Observed band size: 26 kDa

false

• Sanger seq

Supplier Data

Sanger Sequencing - Human TNF knockout THP-1 cell lysate (AB275507)

Homozygous : 52 bp deletion in exon 4