Anti-UBE3A antibody [EPR25059-12] - BSA and Azide free

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-UBE3A antibody [EPR25059-12] - BSA and Azide free (AB290652)

This data was developed using ab290641, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded human cerebrum tissue labeling UBE3A with ab290641 at 1/500 dilution for 30 mins at room temerature, followed by ready to use LeicaDS9800 (BOND™ Polymer Refine Detection). Positive staining can be found in human cerebrum.

The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counter stained with Hematoxylin.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins was used.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is preabsorbed LeicaDS9800 (BOND™ Polymer Refine Detection).

• IHC-Fr

Lab

Immunohistochemistry (Frozen sections) - Anti-UBE3A antibody [EPR25059-12] - BSA and Azide free (AB290652)

Immunohistochemical analysis of rat cerebrum (fresh) frozen sections labelling UBE3A with ab290641 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081) at 1/1000 dilution. Nuclear staining on rat cerebrum is observed. Counter stained with DAPI.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081) at 1/1000 dilution.

This data was developed using ab290641, the same antibody clone in a different buffer formulation.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-UBE3A antibody [EPR25059-12] - BSA and Azide free (AB290652)

This data was developed using ab290641, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded rat cerebrum tissue labeling UBE3A with ab290641 at 1/2000 dilution for 30 mins at room temerature, followed by ready to use LeicaDS9800 (BOND™ Polymer Refine Detection). Positive staining can be found in rat cerebrum (PMID : 22787151).

The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counter stained with Hematoxylin.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins was used.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is preabsorbed LeicaDS9800 (BOND™ Polymer Refine Detection).

• WB

Lab

Western blot - Anti-UBE3A antibody [EPR25059-12] - BSA and Azide free (AB290652)

This data was developed using ab290641, the same antibody clone in a different buffer formulation.

Western blot : Rabbit Monoclonal[EPR25059-12] to UBE3A ab290641 staining at 1/1000 dilution, shown in green; Mouse anti GAPDH (ab8245) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 100 kDa in Wild-type U-87 MG cell lysates with a truncated band observed in UBE3A knockout U-87 MG ab306798 cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3pc 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 800CW & Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-UBE3A antibody [EPR25059-12] (<a href='/en-us/products/primary-antibodies/ube3a-antibody-epr25059-12-ab290641'>ab290641</a>) at 1/1000 dilution

Lane 1:

Wild-type U-87 MG at 20 µg

Lane 2:

Western blot - Human UBE3A knockout U-87 MG cell line (<a href='/en-us/products/cell-lines/human-ube3a-knockout-u-87-mg-cell-line-ab306798'>ab306798</a>) at 20 µg

Lane 2:

knockout U-87 MG at 20 µg

Lane 3:

Human Brain at 20 µg

Lane 4:

K562 at 20 µg

Lane 5:

T-47D at 20 µg

Secondary

Lanes 1 - 5:

Goat anti-Rabbit 800CW at 1/20000 dilution

Lanes 1 - 5:

Goat anti-Mouse 680RD at 1/20000 dilution

Predicted band size: 101 kDa

Observed band size: 100 kDa,36 kDa

false

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-UBE3A antibody [EPR25059-12] - BSA and Azide free (AB290652)

This data was developed using ab290641, the same antibody clone in a different buffer formulation.

Flow cytometry analysis of 4% paraformaldehyde fixed and permeabilized with 90% methanol RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) cells labelling UBE3A with ab290641 at 1/500 dilution (Red) compared with isotype control ab172730 (Black) and unlabelled control cells without incubation whith primary and secondary antibody (Blue).

ab150081 at 1/2000 dilution was used as secondary antibody.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-UBE3A antibody [EPR25059-12] - BSA and Azide free (AB290652)

This data was developed using ab290641, the same antibody clone in a different buffer formulation.

Flow cytometry analysis of 4% paraformaldehyde fixed and permeabilized with 90% methanol HeLa (Human cervix adenocarcinoma epithelial cell) cells labelling UBE3A with ab290641 at 1/500 dilution (Red) compared with isotype control ab172730 (Black) and unlabelled control cells without incubation whith primary and secondary antibody (Blue).

ab150081 at 1/2000 dilution was used as secondary antibody.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-UBE3A antibody [EPR25059-12] - BSA and Azide free (AB290652)

This data was developed using ab290641, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded mouse cerebrum tissue labeling UBE3A with ab290641 at 1/2000 dilution for 30 mins at room temerature, followed by ready to use LeicaDS9800 (BOND™ Polymer Refine Detection). Positive staining can be found in mouse cerebrum (PMID : 22787151).

The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counter stained with Hematoxylin.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins was used.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is preabsorbed LeicaDS9800 (BOND™ Polymer Refine Detection).

• IHC-Fr

Lab

Immunohistochemistry (Frozen sections) - Anti-UBE3A antibody [EPR25059-12] - BSA and Azide free (AB290652)

Immunohistochemical analysis of mouse cerebrum (fresh) frozen sections labelling UBE3A with ab290641 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081) at 1/1000 dilution. Nuclear staining on mouse cerebrum is observed. Counter stained with DAPI.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081) at 1/1000 dilution.

This data was developed using ab290641, the same antibody clone in a different buffer formulation.

• WB

Supplier Data

Western blot - Anti-UBE3A antibody [EPR25059-12] - BSA and Azide free (AB290652)

This data was developed using ab290641, the same antibody clone in a different buffer formulation.

Blocking and diluting buffers : 5% NFDM/TBST.

Exposure time :

Lane 1, 2, 8 : 3 minutes
Lane 3-7 : 26 seconds

Generation of the ~50kDa degraded fragment can be inhibited/reduced by MG-132 treatment (lanes 5 and 7).

All lanes:

Western blot - Anti-UBE3A antibody [EPR25059-12] (<a href='/en-us/products/primary-antibodies/ube3a-antibody-epr25059-12-ab290641'>ab290641</a>) at 1/1000 dilution

Lane 1:

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

Lane 2:

HEK-293 (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 3:

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

Lane 4:

PC-12 (rat adrenal gland pheochromocytoma) whole cell lysate at 20 µg

Lane 5:

PC-12 treated with 10 µM MG-132 for 4 hours, whole cell lysate at 20 µg

Lane 6:

RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg

Lane 7:

RAW264. treated with 10 µM MG-132 for 4 hours, whole cell lysate at 20 µg

Lane 8:

Rat brain tissue lysate at 20 µg

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

Predicted band size: 100 kDa

Observed band size: 100 kDa

false