Anti-UBE2I / UBC9 antibody [EP2938Y]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-UBE2I / UBC9 antibody [EP2938Y] (AB75854)

Immunocytochemistry/ Immunofluorescence analysis of Jurkat (human T cell leukemia T lymphocyte) cells labeling UBE2I / UBC9 with purified ab75854 at 1/100 dilution (10 μg/mL). Cells were fixed in 100% Methanol. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1/200 (2.5 μg/mL). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1/1000 (2 μg/mL) dilution. DAPI (blue) was used as nuclear counterstain. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1/200 (2.5 μg/mL) was used as the secondary antibody only control.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-UBE2I / UBC9 antibody [EP2938Y] (AB75854)

ab75854, at 1/100 dilution, staining UBE2I / UBC9 in human brain, by Immunohistochemistry using formalin-fixed, paraffin-embedded tissue.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-UBE2I / UBC9 antibody [EP2938Y] (AB75854)

Overlay histogram showing HepG2 cells stained with ab75854 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab75854, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

• IP

Unknown

Immunoprecipitation - Anti-UBE2I / UBC9 antibody [EP2938Y] (AB75854)

UBE2I / UBC9 was immunoprecipitated using 0.5mg Jurkat whole cell extract, 5µg of Rabbit polyclonal to UBE2I / UBC9 and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10min, Jurkat whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70^oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab75854.
Secondary : Clean blot (HRP conjugate) at 1/1000 dilution.
Band : 18kDa : UBE2I / UBC9; non specific - 60kDa : We are unsure as to the identity of this extra band.

All lanes:

Immunoprecipitation - Anti-UBE2I / UBC9 antibody [EP2938Y] (ab75854)

Predicted band size: 18 kDa

false

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-UBE2I / UBC9 antibody [EP2938Y] (AB75854)

Immunohistochemical analysis of paraffin-embedded Mouse cerebrum labelling UBE2I / UBC9 with ab75854 at 1/2000 (0.227 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Nuclear staining on the mouse cerebrum
The section was incubated with ab75854 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 Citrate buffer (pH 6.0, Epitope Retrieval Solution 1) for 20 mins.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-UBE2I / UBC9 antibody [EP2938Y] (AB75854)

Immunohistochemical analysis of paraffin-embedded Rat spleen labelling UBE2I / UBC9 with ab75854 at 1/2000 (0.227 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on the Rat spleen
The section was incubated with ab75854 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 Citrate buffer (pH 6.0, Epitope Retrieval Solution 1) for 20 mins.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-UBE2I / UBC9 antibody [EP2938Y] (AB75854)

Immunohistochemical analysis of paraffin-embedded Rat cerebrum labelling UBE2I / UBC9 with ab75854 at 1/2000 (0.227 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Nuclear staining on the Rat cerebrum
The section was incubated with ab75854 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 Citrate buffer (pH 6.0, Epitope Retrieval Solution 1) for 20 mins.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-UBE2I / UBC9 antibody [EP2938Y] (AB75854)

Immunohistochemical analysis of paraffin-embedded Mouse skeletal mucle with ab75854 at 1/2000 (0.227 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Low expression tissue : Negligible staining on Mouse skeletal muscle.

The section was incubated with aab75854 for 30 mins at room temperature.

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

Counterstained with Hematoxylin.

Heat mediated antigen retrieval was performed with Citrate buffer (pH 6.0, Epitope Retrieval Solution 1) for 20 mins.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-UBE2I / UBC9 antibody [EP2938Y] (AB75854)

Immunohistochemical analysis of paraffin-embedded Rat skeletal mucle with ab75854 at 1/2000 (0.227 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Low expression tissue : Negligible staining on Rat skeletal muscle.

The section was incubated with aab75854 for 30 mins at room temperature.

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

Counterstained with Hematoxylin.

Heat mediated antigen retrieval was performed with Citrate buffer (pH 6.0, Epitope Retrieval Solution 1) for 20 mins.

• WB

Unknown

Western blot - Anti-UBE2I / UBC9 antibody [EP2938Y] (AB75854)

All lanes:

Western blot - Anti-UBE2I / UBC9 antibody [EP2938Y] (ab75854) at 1/10000 dilution

Lane 1:

U937 cell lysate at 10 µg

Lane 2:

HeLa cell lysate at 10 µg

Lane 3:

Jurkat cell lysate at 10 µg

Lane 4:

HUVEC cell lysate at 10 µg

Secondary

All lanes:

Goat anti-rabbit HRP at 1/1000 dilution

Predicted band size: 18 kDa

Observed band size: 18 kDa

false

• WB

Supplier Data

Western blot - Anti-UBE2I / UBC9 antibody [EP2938Y] (AB75854)

Diluting and blocking buffer : 5% NFDM/TBST 5% NFDM/TBST

Low expression control : mouse skeletal muscle, rat parathyroid. rat skeletal muscle.

In Western blot, anti-GAPDH antibody (ab181602) loading control staining at 1/200000 dilution.

All lanes:

Western blot - Anti-UBE2I / UBC9 antibody [EP2938Y] (ab75854) at 1/1000 dilution

Lane 1:

Mouse brain tissue lysate at 20 µg

Lane 2:

Mouse spleen tissue lysate at 20 µg

Lane 3:

Mouse skeletal muscle tissue lysate at 20 µg

Lane 4:

Rat brain tissue lysate at 20 µg

Lane 5:

Rat spleen tissue lysate at 20 µg

Lane 6:

Rat parathyroid tissue lysate at 20 µg

Lane 7:

Rat skeletal muscle tissue lysate at 20 µg

Lane 8:

U-937 (human histiocytic lymphoma monocyte) whole cell 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 1/20000 dilution

Predicted band size: 18 kDa

Observed band size: 18 kDa

false

Exposure time: 3s