Human TNFRSF1A (TNF Receptor I) knockout HeLa cell lysate

5 Images

• WB

Lab

Western blot - Human TNFRSF1A (TNF Receptor I) knockout HeLa cell lysate (AB257751)

All lanes : Anti-TNF Receptor I antibody [EPR23742-65] (ab259817) at 1/1000 dilution

Lane 1 : Wild-type HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 2 : TNFRSF1A (TNF Receptor I) knockout HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 3 : A549 (human lung carcinoma epithelial cell) whole cell lysate

Lysates/proteins at 20 μg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (IRDye® 800CW) (ab216773) and Goat Anti-Mouse IgG H&L (IRDye® 680RD) (ab216776) at 1/10000 dilution

Predicted band size : 50 kDa
Observed band size : 50-70 kDa

Blocking and diluting buffer and concentration : 3% NFDM/TBST

Lanes 1-3 : Merged signal (red and green). Green - ab259817 observed at 51 kDa. Red - loading control ab8245 (Mouse monoclonal [6C5] to GAPDH) observed at 36 kDa.

ab259817 Anti-TNF Receptor I antibody [EPR23742-65] was shown to specifically react with TNF Receptor I in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265972 (knockout cell lysate ab257751) was used. Wild-type and TNF Receptor I knockout samples were subjected to SDS-PAGE. ab259817 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at 4°C overnight at 1 in 1000 dilution and 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 10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-TNF Receptor I antibody [EPR23742-65] (<a href='/en-us/products/primary-antibodies/tnf-receptor-i-antibody-epr23742-65-ab259817'>ab259817</a>) at 1/1000 dilution

Lane 1:

Wild-type HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

TNFRSF1A (TNF Receptor I) knockout HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

Western blot - Human TNFRSF1A (TNF Receptor I) knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-tnfrsf1a-tnf-receptor-i-knockout-hela-cell-line-ab265972'>ab265972</a>)

Lane 3:

A549 (human lung carcinoma epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG H&L (IRDye&reg; 800CW) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) and Goat Anti-Mouse IgG H&L (IRDye&reg; 680RD) (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/10000 dilution

Predicted band size: 50 kDa

Observed band size: 50-70 kDa

false

• WB

Lab

Western blot - Human TNFRSF1A (TNF Receptor I) knockout HeLa cell lysate (AB257751)

Lane 1 : Wild-type HeLa cell lysate 20 μg
Lane 2 : HeLa cell lysate 20 μg
False colour image of Western blot : Anti-TNFRSF1A antibody staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, the antibody was shown to bind specifically to TNFRSF1A. A band was observed at 33/63 kDa in wild-type HeLa cell lysates with no signal observed at this size in TNFRSF1A knockout cell line ab265972 (knockout cell lysate ab257751). To generate this image, wild-type and TNFRSF1A knockout HeLa cell lysates were analysed. First, 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:

Anti-TNFRSF1A antibody at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human TNFRSF1A (TNF Receptor I) knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-tnfrsf1a-tnf-receptor-i-knockout-hela-cell-line-ab265972'>ab265972</a>)

false

• WB

Lab

Western blot - Human TNFRSF1A (TNF Receptor I) knockout HeLa cell lysate (AB257751)

Lane 1 : Human wild-type HeLa cell lysate
Lane 2 : Human TNFRSF1A knockout HeLa cell lysate
Lane 3 : Human brain cell lysate
Lane 4 : HL-60 cell lysate

Lanes 1-4 : Merged signal (red and green). Green - anti-TNF-R1 Rabbit monoclonal antibody observed at 51 kDa. Red - loading control, ab8245 observed at 36 kDa.

Anti-TNF-R1 Rabbit monoclonal antibody and anti-GPADH antibody [6C5] - Loading control (ab8245) were incubated overnight at 4^oC at a 1/1000 and 1/20000 dilution respectively before imaging. Blots were developed with Goat anti-Rabbig IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 10,000 dilution for 1 hour at room temperature before imaging.

All lanes:

Anti-TNF-R1 Rabbit monoclonal antibody at 1/1000 dilution

Lane 1:

Human wild-type HeLa cell lysate

Lane 2:

TNFRSF1A knockout HeLa cell lysate

Lane 2:

Western blot - Human TNFRSF1A (TNF Receptor I) knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-tnfrsf1a-tnf-receptor-i-knockout-hela-cell-line-ab265972'>ab265972</a>)

Lane 3:

Human brain cell lysate

Lane 4:

HL-60 cell lysate

false

• Sanger seq

Unknown

Sanger Sequencing - Human TNFRSF1A (TNF Receptor I) knockout HeLa cell lysate (AB257751)

Allele-1 : 2 bp deletion in exon 2

• Sanger seq

Unknown

Sanger Sequencing - Human TNFRSF1A (TNF Receptor I) knockout HeLa cell lysate (AB257751)

Allele-2 : 1 bp deletion in exon 2