Anti-DR4 antibody [EPR28152-57]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-DR4 antibody [EPR28152-57] (AB312848)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HCT 116 (human colorectal carcinoma epithelial cell) cells labelling DR4 with ab312848 at 1/50 (9.72 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2 ug/ml) dilution (Green). Confocal image showing membranous and weak cytoplasmic staining in HCT 116 cell line. Negative control : 293T. Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8). ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 (2.5ug/ml) dilution (Red). The Nuclear counterstain was DAPI (Blue). Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 ug/ml) dilution.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-DR4 antibody [EPR28152-57] (AB312848)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized TNFRSF10A (DR4) knockout HeLa line (Left) / Parental HeLa cell line (Right) cells labelling DR4 with ab312848 at 1/500 dilution (0.1 ug)/Red (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

• WB

Lab

Western blot - Anti-DR4 antibody [EPR28152-57] (AB312848)

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

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/1000000 dilution.

In Western blot, Anti-Myc tag antibody [9E10] - Loading Control (ab32) staining at 1/10000 dilution.

In Western blot, Anti-6X His tag® antibody [EPR20547] - Loading Control (ab213204) staining at 1/10000 dilution.

This antibody does not cross react with DR5 and DcR2.

All lanes:

Western blot - Anti-DR4 antibody [EPR28152-57] (ab312848) at 1/1000 dilution

Lane 1:

293T (Human embryonic kidney epithelial cell) transfected with an empty vector (vector control), containing a myc-His- tag® whole cell lysate at 20 µg

Lane 2:

293T transfected with human DR4 expression vector containing a myc- His-tag®, whole cell lysate at 20 µg

Lane 3:

293T transfected with human DcR2 expression vector containing a myc- His-tag®, whole cell lysate at 48 µg

Lane 4:

Recombinant human DR5 fragment protein containing a His-tag®, whole cell lysate at 10 ng

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 48 kDa

false

Exposure time: 3s

• WB

Supplier Data

Western blot - Anti-DR4 antibody [EPR28152-57] (AB312848)

Blocking and diluting buffer and concentration : 5% NFDM/TBST Performed under reducing conditions. In Western blot, ab312848 was shown to bind specifically to TNFRSF10A (DR4). A band was observed at 45 kDa in wild-type Hela cell lysates whereas loss of signal was observed in the TNFRSF10A (DR4) knockout cell line ab264687 (knockout cell lysate ab258726). In Western blot, anti- Vinculin antibody (ab129002) loading control staining at 1/10000 dilution. Exposure time : 15 seconds

All lanes:

Western blot - Anti-DR4 antibody [EPR28152-57] (ab312848) at 1/1000 dilution

Lane 1:

Wild-type HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 50 µg

Lane 2:

Western blot - Human TNFRSF10A (DR4) knockout HeLa cell lysate (<a href='/en-us/products/cell-lysates/human-tnfrsf10a-dr4-knockout-hela-cell-lysate-ab258726'>ab258726</a>) at 50 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Observed band size: 45 kDa

false

Exposure time: 15s

• WB

Supplier Data

Western blot - Anti-DR4 antibody [EPR28152-57] (AB312848)

Blocking and diluting buffer and concentration : 5% NFDM/TBST Negative control : 293T, Fadu. In Western blot, anti-GAPDH antibody (ab181602) loading control staining at 1/200000 dilution. Exposure time : 15 seconds

All lanes:

Western blot - Anti-DR4 antibody [EPR28152-57] (ab312848) at 1/1000 dilution

Lane 1:

HCT 116 (human colorectal carcinoma epithelial cell) whole cell lysate at 50 µg

Lane 2:

Fadu (human pharyeal carcinoma epithelial cell) whole cell lysate at 50 µg

Lane 3:

293T (human embryonic kidney epithelial cell) whole cell lysate at 50 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Observed band size: 45 kDa

false

Exposure time: 15s