Anti-Ubiquitin antibody [EPR8830]

• WB

Unknown

Western blot - Anti-Ubiquitin antibody [EPR8830] (AB134953)

Blocking and diluting buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-Ubiquitin antibody [EPR8830] (ab134953) at 0.7 µg/mL

Lane 1:

Rat brain lysate at 20 µg

Lane 2:

Mouse kidney lysate at 20 µg

Lane 3:

Rat kidney 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: 26 kDa

Observed band size: 8 kDa

false

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Ubiquitin antibody [EPR8830] (AB134953)

Immunohistochemical analysis of paraffin embedded Human breast carcinoma tissue labelling Ubiquitin with ab134953 at 1/250 dilution

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Ubiquitin antibody [EPR8830] (AB134953)

Immunofluorescent staining of JAR cells labelling Ubiquitin using ab134953 at 1/250 dilution

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Ubiquitin antibody [EPR8830] (AB134953)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human bladder carcinoma tissue sections labeling Ubiquitin with Purified ab134953 at 1 : 800 dilution (0.9 μg/ml). Heat mediated antigen retrieval was performed using EDTA Buffer, pH 9.0. Tissue was counterstained with Hematoxylin. ab97051 Goat Anti-Rabbit IgG H&L (HRP) secondary antibody was used at 1 : 500 dilution. PBS instead of the primary antibody was used as the negative control.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Ubiquitin antibody [EPR8830] (AB134953)

Immunocytochemistry/ Immunofluorescence analysis of JAR (Human placenta choriocarcinoma cell line) cells labeling Ubiquitin with Purified ab134953 at 1 : 100 dilution (7.2μg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1 : 200 (2.5 μg/ml). ab150077 Goat anti rabbit IgG(Alexa Fluor^® 488) was used as the secondary antibody at 1 : 1000 dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Ubiquitin antibody [EPR8830] (AB134953)

Overlay histogram showing HepG2 cells stained with ab134953 (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 (ab134953, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. This antibody gave a positive signal in HepG2 cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Ubiquitin antibody [EPR8830] (AB134953)

Intracellular Flow Cytometry analysis of JAR (Human placenta choriocarcinoma cell line) cells labeling Ubiquitin with purified ab134953 at 1/70 dilution (10 ug/ml) (red). Cells were fixed with 4% Paraformaldehyde. A Goat anti rabbit IgG (Alexa Fluor^® 488) secondary antibody was used at 1/2000 dilution. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Ubiquitin antibody [EPR8830] (AB134953)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Rat liver tissue sections labeling Ubiquitin with Purified ab134953 at 1 : 800 dilution (0.9 μg/ml). Heat mediated antigen retrieval was performed using EDTA Buffer, pH 9.0. Tissue was counterstained with Hematoxylin. ab97051 Goat Anti-Rabbit IgG H&L (HRP)
secondary antibody was used at 1 : 500 dilution. PBS instead of the primary antibody was used as the negative control.

• IHC-P

AbReview38032****

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Ubiquitin antibody [EPR8830] (AB134953)

ab134953 staining Ubiquitin in Mouse embryo tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde and blocked with 1% FBS/BSA for 1 hour at room temperature; antigen retrieval was by heat mediation in citrate buffer, pH6. Samples were incubated with primary antibody (1/100 in 1% FBS/BSA) for 16 hours at 4°C. An undiluted HRP-conjugated Goat anti-rabbit IgG polyclonal was used as the secondary antibody.

This image is courtesy of an anonymous Abreview.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Ubiquitin antibody [EPR8830] (AB134953)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Mouse liver tissue sections labeling Ubiquitin with Purified ab134953 at 1 : 800 dilution (0.9 μg/ml). Heat mediated antigen retrieval was performed using EDTA Buffer, pH 9.0. Tissue was counterstained with Hematoxylin. ab97051 Goat Anti-Rabbit IgG H&L (HRP)
secondary antibody was used at 1 : 500 dilution. PBS instead of the primary antibody was used as the negative control.

• WB

Lab

Western blot - Anti-Ubiquitin antibody [EPR8830] (AB134953)

This blot was produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 35 minutes before being transferred onto a nitrocellulose membrane at 30V for 70 minutes .ab134953 and ab8245 (loading control to GAPDH) were diluted 1/200 and 1/10 000 respectively and incubated overnight at 4°C. Blots were developed with goat anti-rabbit IgG (H + L) and goat anti-mouse IgG (H + L) secondary antibodies at 1/10 000 dilution for 1 h at room temperature before imaging using the Licor Odyssey CLx.

All lanes:

Western blot - Anti-Ubiquitin antibody [EPR8830] (ab134953) at 1/200 dilution

Lane 1:

MCF-7 Whole cell lysate at 20 µg

Lane 2:

MCF-7 Whole cell lysate + MG-132 (50 uM 90 min) at 20 µg

Lane 3:

Mouse Brain at 20 µg

Predicted band size: 26 kDa

false

• WB

Unknown

Western blot - Anti-Ubiquitin antibody [EPR8830] (AB134953)

Note : Ubiquitin exists as monomeric ubiquitin (8 kDa) or attached to other proteins.

All lanes:

Western blot - Anti-Ubiquitin antibody [EPR8830] (ab134953) at 1/1000 dilution

Lane 1:

293T cell lysate at 10 µg

Lane 2:

HepG2 cell lysate at 10 µg

Lane 3:

HeLa cell lysate at 10 µg

Lane 4:

JAR cell lysate at 10 µg

Secondary

All lanes:

HRP labelled goat anti rabbit at 1/2000 dilution

Predicted band size: 26 kDa

false

• WB

Supplier Data

Western blot - Anti-Ubiquitin antibody [EPR8830] (AB134953)

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

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

In Western blot, Anti-Ubiquitin antibody [EPR8830] (ab134953) staining at 1/1000 dilution.

All lanes:

Western blot - Anti-Ubiquitin (phospho S65) antibody [30H3/30K1] - Rat IgG2a (Chimeric) (<a href='/en-us/products/primary-antibodies/ubiquitin-phospho-s65-antibody-30h3-30k1-rat-igg2a-chimeric-ab320096'>ab320096</a>) at 1/1000 dilution

Lane 1:

Untreated PC-3 (human prostate adenocarcinoma epithelial cell) whole cell lysate (untreated membrane) at 20 µg

Lane 2:

PC-3 treated with 30 μM CCCP (<a href='/en-us/products/biochemicals/cccp-mitochondrial-oxidative-phosphorylation-uncoupler-ab141229'>ab141229</a>) for 2 hours, whole cell lysate (untreated membrane) at 20 µg

Lane 3:

Untreated PC-3 (human prostate adenocarcinoma epithelial cell) whole cell lysate (Alkaline phosphatase treated membrane) at 20 µg

Lane 4:

PC-3 treated with 30 μM CCCP (<a href='/en-us/products/biochemicals/cccp-mitochondrial-oxidative-phosphorylation-uncoupler-ab141229'>ab141229</a>) for 2 hours, whole cell lysate (Alkaline phosphatase treated membrane) at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rat IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rat-igg-h-l-hrp-ab205720'>ab205720</a>) at 1/10000 dilution

Observed band size: 8-200 kDa,36 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-Ubiquitin antibody [EPR8830] (AB134953)

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

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

In Western blot, Anti-Ubiquitin antibody [EPR8830] (ab134953) staining at 1/1000 dilution.

All lanes:

Western blot - Anti-Ubiquitin (phospho S65) antibody [30H3/30K1] - Rat IgG2a (Chimeric) (<a href='/en-us/products/primary-antibodies/ubiquitin-phospho-s65-antibody-30h3-30k1-rat-igg2a-chimeric-ab320096'>ab320096</a>) at 1/1000 dilution

Lane 1:

Untreated Wild-type 293T (human embryonic kidney epithelial cell) whole cell lysate (untreated membrane) at 20 µg

Lane 2:

293T treated with 20 μM CCCP (<a href='/en-us/products/biochemicals/cccp-mitochondrial-oxidative-phosphorylation-uncoupler-ab141229'>ab141229</a>) for 8 hours, whole cell lysate (untreated membrane) at 20 µg

Lane 3:

Untreated PINK1 knockout HEK-293T whole cell lysate (untreated membrane) at 20 µg

Lane 4:

PINK1 knockout HEK-293T treated with 20 μM CCCP (<a href='/en-us/products/biochemicals/cccp-mitochondrial-oxidative-phosphorylation-uncoupler-ab141229'>ab141229</a>) for 8 hours, whole cell lysate (untreated membrane) at 20 µg

Lane 5:

Untreated Wild-type 293T (human embryonic kidney epithelial cell) whole cell lysate (Alkaline phosphatase treated membrane) at 20 µg

Lane 6:

293T treated with 20 μM CCCP (<a href='/en-us/products/biochemicals/cccp-mitochondrial-oxidative-phosphorylation-uncoupler-ab141229'>ab141229</a>) for 8 hours, whole cell lysate (Alkaline phosphatase treated membrane) at 20 µg

Lane 7:

Untreated PINK1 knockout HEK-293T whole cell lysate (Alkaline phosphatase treated membrane) at 20 µg

Lane 8:

PINK1 knockout HEK-293T treated with 20 μM CCCP (<a href='/en-us/products/biochemicals/cccp-mitochondrial-oxidative-phosphorylation-uncoupler-ab141229'>ab141229</a>) for 8 hours, whole cell lysate (Alkaline phosphatase treated membrane) at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rat IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rat-igg-h-l-hrp-ab205720'>ab205720</a>) at 1/10000 dilution

Observed band size: 8-200 kDa,36 kDa

false

Exposure time: 92s

• WB

Unknown

Western blot - Anti-Ubiquitin antibody [EPR8830] (AB134953)

Blocking and diluting buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-Ubiquitin antibody [EPR8830] (ab134953) at 0.1 µg/mL

Lane 1:

JAR (Human placenta choriocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

293 (Human embryonic kidney epithelial cell) 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: 26 kDa

Observed band size: 8 kDa

false

• WB

Supplier Data

Western blot - Anti-Ubiquitin antibody [EPR8830] (AB134953)

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

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

In Western blot, Anti-Ubiquitin antibody [EPR8830] (ab134953) staining at 1/1000 dilution.

All lanes:

Western blot - Anti-Ubiquitin (phospho S65) antibody [30H3/30K1] - Rat IgG2a (Chimeric) (<a href='/en-us/products/primary-antibodies/ubiquitin-phospho-s65-antibody-30h3-30k1-rat-igg2a-chimeric-ab320096'>ab320096</a>) at 1/1000 dilution

Lane 1:

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

Lane 2:

MEF treated with 5 μM Valinomycin for 24 hours whole cell lysate (untreated membrane) at 20 µg

Lane 3:

Untreated MEF (mouse embryo fibroblast) whole cell lysate (Alkaline phosphatase treated membrane) at 20 µg

Lane 4:

MEF treated with 5 μM Valinomycin for 24 hours whole cell lysate (Alkaline phosphatase treated membrane) at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rat IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rat-igg-h-l-hrp-ab205720'>ab205720</a>) at 1/10000 dilution

Observed band size: 8-200 kDa,36 kDa

false

Exposure time: 92s