Recombinant Anti-UBE2C antibody [EPR23165-31] (ab252940)

False colour image of Western blot: Anti-UBE2C antibody [EPR23165-31] 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, ab252940 was shown to bind specifically to UBE2C. A band was observed at 20 kDa in wild-type HeLa cell lysates with no signal observed at this size in UBE2C knockout cell line ab265032 (knockout cell lysate ab257775). To generate this image, wild-type and UBE2C 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 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) 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.

Immunohistochemical analysis of paraffin-embedded human gastric cancer tissue labeling UBE2C with ab252940 at 1/500 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Cytoplasm and nuclear staining in proliferative cells of human gastric cancer is observed (PMID:15208666, 27349176). The section was incubated with ab252940 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with hematoxylin.

Secondary antibody only control: Ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20mins.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) cells labeling UBE2C with ab252940 at 1/50 dilution, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1/1000 dilution (Green). Confocal image showing nuclear and cytoplasmic staining in partial RAW 264.7 cells. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The nuclear counterstain was DAPI (Blue).

Secondary antibody only control: ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution.

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

The molecular weight observed is consistent with what has been described in the literature (PMID: 27349176, 17217624).

Exposure time: 3 minutes.

Immunohistochemical analysis of paraffin-embedded mouse colon tissue labeling UBE2C with ab252940 at 1/500 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining in proliferative cells of mouse colon is observed (PMID: 15208666). The section was incubated with ab252940 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with hematoxylin.

Secondary antibody only control: Ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20mins.

UBE2C was immunoprecipitated from 0.35 mg NIH:OVCAR-3 (Human ovary adenocarcinoma epithelial cell) whole cell lysate 10µg with ab252940 at 1/30 dilution (2µg in 0.35 mg lysates). Western blot was performed on the immunoprecipitate using ab252940 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1: NIH:OVCAR-3 whole cell lysate 10µg.

Lane 2: ab252940 IP in NIH:OVCAR-3 whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab252940 in NIH:OVCAR-3 whole cell lysate.

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

Exposure time: 30 seconds.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized SW480 (human colorectal adenocarcinoma epithelial cell) cells labeling UBE2C with ab252940 at 1/50 dilution, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1/1000 dilution (Green). Confocal image showing nuclear and cytoplasmic staining in partial SW480 cells. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The nuclear counterstain was DAPI (Blue).

Secondary antibody only control: ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution.

Immunohistochemical analysis of paraffin-embedded rat colon tissue labeling UBE2C with ab252940 at 1/500 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining in proliferative cells of rat colon is observed (PMID: 15208666). The section was incubated with ab252940 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with hematoxylin.

Secondary antibody only control: Ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20mins.

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

The molecular weight observed is consistent with what has been described in the literature (PMID: 27349176, 17217624).

Exposure time: 3 minutes.

Immunohistochemical analysis of paraffin-embedded human colon tissue labeling UBE2C with ab252940 at 1/500 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Cytoplasm and nuclear staining in proliferative cells of human colon is observed (PMID: 15208666). The section was incubated with ab252940 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with hematoxylin.

Secondary antibody only control: Ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20mins.

UBE2C was immunoprecipitated from 0.35 mg SW480 (Human colorectal adenocarcinoma epithelial cell) whole cell lysate 10µg with ab252940 at 1/30 dilution (2µg in 0.35 mg lysates). Western blot was performed on the immunoprecipitate using ab252940 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1: SW480 whole cell lysate 10µg.

Lane 2: ab252940 IP in SW480 whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab252940 in SW480 whole cell lysate.

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

Exposure time: 30 seconds.