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 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).

All lanes : Anti-Ubiquitin antibody [EPR8830] (ab134953) at 0.7 µg/ml (Purified)

Lane 1 : Rat brain lysate
Lane 2 : Mouse kidney lysate
Lane 3 : Rat kidney lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 8 kDa
Observed band size: 8 kDa

Blocking and diluting buffer: 5% NFDM/TBST

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.

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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.

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.

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.

All lanes : Anti-Ubiquitin antibody [EPR8830] (ab134953) at 0.1 µg/ml (Purified)

Lane 1 : JAR (Human placenta choriocarcinoma epithelial cell) whole cell lysate
Lane 2 : 293 (Human embryonic kidney epithelial cell) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 8 kDa
Observed band size: 8 kDa

Blocking and diluting buffer: 5% NFDM/TBST

All lanes : Anti-Ubiquitin antibody [EPR8830] (ab134953) at 1/200 dilution

Lane 1 : MCF-7 Whole cell lysate
Lane 2 : MCF-7 Whole cell lysate + MG-132 (50 uM 90 min)
Lane 3 : Mouse Brain

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 8 kDa

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.

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.

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

All lanes : Anti-Ubiquitin antibody [EPR8830] (ab134953) at 1/1000 dilution

Lane 1 : 293T cell lysate
Lane 2 : HepG2 cell lysate
Lane 3 : HeLa cell lysate
Lane 4 : JAR cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP labelled goat anti rabbit at 1/2000 dilution

Predicted band size: 8 kDa



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

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