Anti-E3 ubiquitin-protein ligase RNF213 antibody [EPR28972-85]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-E3 ubiquitin-protein ligase RNF213 antibody [EPR28972-85] (AB327703)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HC11 (mouse mammary gland epithelial cell) cells labelling C17orf27 with ab327703 at 1/50 (10.14 μg/ml) dilution , followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Confocal image showing cytoplasmic staining in HC11 cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab7291 Anti-alpha Tubulin mouse monoclonal antibody was used to counterstain tubulin at 1/1000 dilution (Magenta). 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 dilution.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-E3 ubiquitin-protein ligase RNF213 antibody [EPR28972-85] (AB327703)

Immunohistochemical analysis of paraffin-embedded (A) Mouse cerebrum (B) RNF213 knockout mouse cerebrum tissue labeling C17orf27 with ab327703 at 1/1000 (0.507 μg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive on endothelial cells in (A) Mouse cerebrum, no staining on (B) RNF213 knockout mouse cerebrum. The primary antibody was incubated for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-E3 ubiquitin-protein ligase RNF213 antibody [EPR28972-85] (AB327703)

Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labeling C17orf27 with ab327703 at 1/1000 (0.507 μg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mouse spleen. The primary antibody was incubated for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-E3 ubiquitin-protein ligase RNF213 antibody [EPR28972-85] (AB327703)

Immunohistochemical analysis of paraffin-embedded Mouse hepatocellular carcinoma tissue labeling C17orf27 with ab327703 at 1/1000 (0.507 μg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mouse hepatocellular carcinoma. The primary antibody was incubated for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• WB

Lab

Western blot - Anti-E3 ubiquitin-protein ligase RNF213 antibody [EPR28972-85] (AB327703)

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

The tissue samples were kindly provided by GemPharmatech. C57BL/6JGpt wildtype mice and E3 ubiquitin-protein ligase RNF213-KO homozygous mice (Strain ID : T011543).

To minimize protein degradation, the fresh lysates in lanes 6-7 were lysed immediately after harvest and then applied to a gel and transfer membrane for Western blotting as soon as possible. Lanes 6-7 of this blot was developed using a high-sensitivity ECL substrate, allowing for the detection of proteins in the mid-femtogram range.

In Western blot, Anti-Vinculin antibody [EPR8185] (ab129002) (1 : 10000) (124KDa).

Exposure time : Lanes 1-3 : 59 seconds; Lanes 4-7 : 180 seconds

All lanes:

Western blot - Anti-E3 ubiquitin-protein ligase RNF213 antibody [EPR28972-85] (ab327703) at 1/1000 dilution

Lane 1:

Wild-type mouse thymus tissue lysate (female) at 20 µg

Lane 2:

E3 ubiquitin-protein ligase RNF213 knockout mouse thymus tissue lysate (female) at 20 µg

Lane 3:

MEF (mouse embryo fibroblast) whole cell lysate at 20 µg

Lane 4:

HC11 (mouse mammary gland epithelial cell) whole cell lysate at 20 µg

Lane 5:

Neuro-2a (mouse neuroblastoma neuroblast) whole cell lysate at 20 µg

Lane 6:

Mouse skeletal muscle tissue lysate at 20 µg

Lane 7:

Mouse spleen tissue lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/ko/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 584 kDa,124 kDa

true

• WB

Lab

Western blot - Anti-E3 ubiquitin-protein ligase RNF213 antibody [EPR28972-85] (AB327703)

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

The lysates were kindly provided by an anonymous collaborator.

Performed under reducing conditions.

In Western blot, ab327703 was shown to bind specifically to E3 ubiquitin-protein ligase RNF213. Target of interest was observed at 584 kD in wild-type MEF cell lysates (lane 1) with no signal observed at this size in the E3 ubiquitin-protein ligase RNF213 knockout cell line (lanes 2).

In Western blot, Anti-Vinculin antibody [EPR8185] (ab129002) (1 : 10000) (124KDa).

All lanes:

Western blot - Anti-E3 ubiquitin-protein ligase RNF213 antibody [EPR28972-85] (ab327703) at 1/1000 dilution

Lane 1:

Wild-type MEF (mouse embryo fibroblast) whole cell lysate at 20 µg

Lane 2:

E3 ubiquitin-protein ligase RNF213 knockout MEF whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/ko/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 584 kDa,124 kDa

false

Exposure time: 180s