Anti-UBE2C antibody

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-UBE2C antibody (AB12290)

ICC/IF image of ab12290 stained human HEK 293 cells. The cells were methanol fixed (5 min), permabilised in 0.1% PBS-Tween (20 min) and incubated with the antibody (ab12290, 1μg/ml) for 1h at room temperature. 1%BSA / 10% normal goat serum / 0.3M glycine was used to block non-specific protein-protein interactions. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red). DAPI was used to stain the cell nuclei (blue). This antibody also gave a positive IF result in HepG2 and MCF7 cells.

• WB

Lab

Western blot - Anti-UBE2C antibody (AB12290)

False colour image of Western blot : Anti-UBE2C 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, ab12290 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 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:

Western blot - Anti-UBE2C antibody (ab12290) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

UBE2C knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human UBE2C knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-ube2c-knockout-hela-cell-line-ab265032'>ab265032</a>)

Lane 3:

WI-38 cell lysate at 20 µg

Lane 4:

K-562 (Human chronic myelogenous leukemia lymphoblast cell line ) whole cell lysate at 20 µg

Lane 5:

SW480 cell lysate at 20 µg

Lane 6:

CACO2 cell lysate at 20 µg

Predicted band size: 20 kDa

Observed band size: 20 kDa

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• WB

Unknown

Western blot - Anti-UBE2C antibody (AB12290)

All lanes:

Western blot - Anti-UBE2C antibody (ab12290) at 1 µg/mL

Lane 1:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg

Lane 2:

Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate at 10 µg

Lane 3:

Jurkat nuclear extract lysate (<a href='/en-us/products/unavailable/jurkat-nuclear-extract-lysate-ab14844'>ab14844</a>) at 10 µg

Lane 4:

A431 (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg

Lane 5:

HEK293 Human embryonic kidney cell line Whole Cell Lysate at 10 µg

Secondary

All lanes:

IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution

Predicted band size: 20 kDa

Observed band size: 20 kDa,55 kDa,70 kDa

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