Anti-Ubiquitin (linkage-specific K48) antibody [EP8589] (ab140601) at 1/1000 dilution + K48-linked-Ub2-7recombinant protein lysate at 15 µg

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

Predicted band size: 77 kDa

Blocking and diluting buffer: 5% NFDM/TBST. This image is produced useing purified ab140601.

Purified ab140601 staining Ubiquitin (linkage-specific K48) in MCF7 (Human breast adenocarcinoma cell line) cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with 4% Paraformaldehyde and permeabilized with 0.1% Triton X-100. Samples were incubated with primary antibody at a dilution of 1/500. A goat anti rabbit IgG (Alexa Fluor® 488) (ab150077) was used as the secondary antibody at a dilution of 1/1000. ab195889 was used as a counterstain for primary antibody ab133645 at 1/2000. DAPI was used as a nuclear counterstain and PBS as a negative control.

Purified ab140601 staining Ubiquitin (linkage-specific K48) in human endometrium carcinoma tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and antigen retrieval was by heat mediation in a EDTA buffer. Samples were incubated with primary antibody at a dilution of 1/250. A goat anti-rabbit IgG H&L (HRP) ab97051 was used as the secondary antibody at a dilution of 1/500.

Negative control 1: PBS in place of primary antibody.

All lanes : Anti-Ubiquitin (linkage-specific K48) antibody [EP8589] (ab140601) at 200 µg

Lane 1 : Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate
Lane 2 : 293 (Human embryonic kidney epithelial cell) whole cell lysate
Lane 3 : Mouse heart lysate
Lane 4 : Rat heart lysate
Lane 5 : C2C12 (Mouse myoblasts myoblast) whole cell lysate
Lane 6 : C6 (Rat glial tumor glial 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: 77 kDa

Blocking and diluting buffer: 5% NFDM/TBST. This image is produced useing purified ab140601.

All lanes : Anti-Ubiquitin (linkage-specific K48) antibody [EP8589] (ab140601) at 1/200 dilution

Lane 1 : PC-12
Lane 2 : C6
Lane 3 : L6
Lane 4 : C2C12
Lane 5 : Neuro-2a
Lane 6 : NIH3T3
Lane 7 : SP2/0
Lane 8 : Raw264.7
Lane 9 : B16-F0

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 77 kDa

Observed band is above 60kDa

All lanes : Anti-Ubiquitin (linkage-specific K48) antibody [EP8589] (ab140601) at 1/200 dilution

Lane 1 : HEK293
Lane 2 : Jurkat

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 77 kDa

Observed band is above 60kDa 

All lanes : Anti-Ubiquitin (linkage-specific K48) antibody [EP8589] (ab140601) at 1/1000 dilution

Lane 1 : K6-linked-Ub2 recombinant protein
Lane 2 : K27-linked-Ub2 recombinant protein
Lane 3 : K29-linked-Ub2 recombinant protein
Lane 4 : K11-linked-Ub2 recombinant protein
Lane 5 : K48-linked-Ub2-7recombinant protein
Lane 6 : K63-linked-Ub2-7 recombinant protein
Lane 7 : K33-linked-Ub2 recombinant protein
Lane 8 : monoubiquitin recombinant protein

Lysates/proteins at 0.01 µg per lane.

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

Predicted band size: 77 kDa
Observed band size: 17-60 kDa why is the actual band size different from the predicted?

ICC/IF image of ab140601 stained MCF-7 cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab140601 at 10µg/ml overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- rabbit (ab96899) IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

Overlay histogram showing HeLa cells stained with ab140601 (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 (ab140601, 1/100 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) (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.