Anti-TNF Receptor I antibody [EPR23742-65]

• WB

Lab

Western blot - Anti-TNF Receptor I antibody [EPR23742-65] (AB259817)

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] (ab259817) 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 - Anti-TNF Receptor I antibody [EPR23742-65] (AB259817)

Blocking and diluting buffer and concentration : 5% NFDM/TBST

The difference in target band size observed in HeLa and HepG2 cell lysates is caused by glycosylation.

Negative control : Raji (PMID : 1966549; PMID : 2158104).

Exposure time : 81 seconds

All lanes:

Western blot - Anti-TNF Receptor I antibody [EPR23742-65] (ab259817) at 1/1000 dilution

Lane 1:

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

Lane 2:

HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

Raji (human Burkitt's lymphoma B lymphocyte) whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/50000 dilution

Predicted band size: 50 kDa

Observed band size: 50-70 kDa

false