Anti-Ubiquitin (linkage-specific K48) antibody [EP8589]

• WB

Lab

Western blot - Anti-Ubiquitin (linkage-specific K48) antibody [EP8589] (AB140601)

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

All lanes:

Western blot - Anti-Ubiquitin (linkage-specific K48) antibody [EP8589] (ab140601) at 1/1000 dilution

All lanes:

K48-linked-Ub2-7recombinant protein lysate at 15 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,35 kDa,38 kDa,40 kDa,58 kDa,79 kDa

Observed band size: 26 kDa,38 kDa,39 kDa,42 kDa,78 kDa

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• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Ubiquitin (linkage-specific K48) antibody [EP8589] (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.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Ubiquitin (linkage-specific K48) antibody [EP8589] (AB140601)

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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Ubiquitin (linkage-specific K48) antibody [EP8589] (AB140601)

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.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Ubiquitin (linkage-specific K48) antibody [EP8589] (AB140601)

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.

• IHC

Lab

Immunohistochemistry - Anti-Ubiquitin (linkage-specific K48) antibody [EP8589] (AB140601)

Immunohistochemical analysis of formalin fixed paraffin embedded human endometrial carcinoma labelling Ubiquitin (linkage-specific K48) with ab140601 at a concentration of 1µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH 8.5 for 32 mins.

ab140601 anti-Ubiquitin (linkage-specific K48) [EP8589] was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual)

• WB

Supplier Data

Western blot - Anti-Ubiquitin (linkage-specific K48) antibody [EP8589] (AB140601)

Observed band is above 60kDa

All lanes:

Western blot - Anti-Ubiquitin (linkage-specific K48) antibody [EP8589] (ab140601) at 1/200 dilution

Lane 1:

HEK293 at 10 µg

Lane 2:

Jurkat at 10 µg

Secondary

All lanes:

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

Predicted band size: 26 kDa

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• WB

Lab

Western blot - Anti-Ubiquitin (linkage-specific K48) antibody [EP8589] (AB140601)

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

All lanes:

Western blot - Anti-Ubiquitin (linkage-specific K48) antibody [EP8589] (ab140601) at 200 µg

Lane 1:

Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate at 20 µg

Lane 2:

293 (Human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 3:

Mouse heart lysate at 20 µg

Lane 4:

Rat heart lysate at 20 µg

Lane 5:

C2C12 (Mouse myoblasts myoblast) whole cell lysate at 20 µg

Lane 6:

C6 (Rat glial tumor glial 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

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• WB

Supplier Data

Western blot - Anti-Ubiquitin (linkage-specific K48) antibody [EP8589] (AB140601)

Observed band is above 60kDa

All lanes:

Western blot - Anti-Ubiquitin (linkage-specific K48) antibody [EP8589] (ab140601) at 1/200 dilution

Lane 1:

PC-12 at 20 µg

Lane 2:

C6 at 20 µg

Lane 3:

L6 at 20 µg

Lane 4:

C2C12 at 20 µg

Lane 5:

Neuro-2a at 20 µg

Lane 6:

NIH3T3 at 20 µg

Lane 7:

SP2/0 at 20 µg

Lane 8:

Raw264.7 at 20 µg

Lane 9:

B16-F0 at 20 µg

Secondary

All lanes:

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

Predicted band size: 26 kDa

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• WB

Unknown

Western blot - Anti-Ubiquitin (linkage-specific K48) antibody [EP8589] (AB140601)

All lanes:

Western blot - Anti-Ubiquitin (linkage-specific K48) antibody [EP8589] (ab140601) at 1/1000 dilution

Lane 1:

K6-linked-Ub2 recombinant protein at 0.01 µg

Lane 2:

K27-linked-Ub2 recombinant protein at 0.01 µg

Lane 3:

K29-linked-Ub2 recombinant protein at 0.01 µg

Lane 4:

K11-linked-Ub2 recombinant protein at 0.01 µg

Lane 5:

K48-linked-Ub2-7recombinant protein at 0.01 µg

Lane 6:

K63-linked-Ub2-7 recombinant protein at 0.01 µg

Lane 7:

K33-linked-Ub2 recombinant protein at 0.01 µg

Lane 8:

monoubiquitin recombinant protein at 0.01 µg

Secondary

All lanes:

HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 26 kDa

Observed band size: 17-60 kDa

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• WB

CiteAb

Western blot - Anti-Ubiquitin (linkage-specific K48) antibody [EP8589] (AB140601)

Ubiquitin (linkage-specific K48) western blot using anti-Ubiquitin (linkage-specific K48) antibody [EP8589] ab140601. Publication image and figure legend from Sambri, I., D'Alessio, R., et al., 2017, EMBO Mol Med, PubMed 27881461.

ab140601 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab140601 please see the product overview.

Lysosomal-driven deregulation of α-synuclein and CSPα degradationWB analysis of LC3 and p62 (an autophagy substrate) was performed on WT and MPS-IIIA brain samples at the indicated ages. WB quantitation is shown.α-Synuclein was immunoblotted in WT and MPS-IIIA in both total and synaptosomal brain fractions at the indicated ages after sequential extraction with detergents with increased strength. Soluble (Sol.), lowly insoluble (L. Insol.), and highly insoluble (H. Insol.) forms correspond to the protein solubilized in Triton X-100, 10% SDS and 8 M urea, respectively.Co-immunofluorescence analysis of α-synuclein with SMI-32 in WT and MPS-IIIA hippocampal neurons (DIV14). α-Synuclein synaptic puncta present in a neurite tract of 10 μm is shown in a representative enlarged image. Quantification of α-synuclein synaptic puncta was calculated from 30 different enlarged images.Confocal analysis of α-synuclein (green) and LAMP1 (red) in WT and MPS-IIIA hippocampal neurons (DIV14). Enlarged merge images are also shown. Co-localization was quantified using the MCC (ImageJ) and displayed as percentage (MCC × 100) of α-synuclein co-localizing with LAMP1 (15 different images taken from 4 to 5 coverslips for each group).CSPα was immunoblotted in WT and MPS-IIIA total brain lysates at the indicated ages after sequential extraction with detergents with increased strength as in (B).Co-immunofluorescence analysis of CSPα and SMI-32 in DIV14 hippocampal neurons. CSPα synaptic puncta was quantified as in (C).CSPα protein levels were evaluated by immunoblot analysis in WT and MPS-IIIA hippocampal neurons (DIV14) at different times after cycloheximide treatment and expressed as percentage of remaining protein at T0 (100%). The proteasome was inhibited as indicated. WB quantification is shown.Palmitoylation-dependent shift in the molecular weight of CSPα was evaluated in WT and MPS-IIIA brain samples at the indicated ages by immunoblot CSPα in boiled samples prepared without exposure to sulfhydryl agents (β-mercaptoethanol or dithiothreitol).The protein levels of RPN10 (the regulatory subunit of 26S proteasome) and ubiquitinated proteins formed by Lys48 (K48) residue linkage (involved in protein degradation via the proteasome) were evaluated in WT and MPS-IIIA brain samples at the indicated ages by WB analysis. WB quantitation is shown.Proteasome activity was evaluated by measuring the chymotrypsin-like activity in WT and MPS-IIIA mouse brain samples at different ages. Proteasome activity was expressed as percentage of WT activity.Data information : Data are means ± s.e.m.; N = 3 (biological triplicate) in WB quantitations. *p < 0.05, **p < 0.001, Student's t-test : MPS-IIIA at each age vs. WT (A, I, J); MPS-IIIA vs. WT (C, D, F); either WT or MPS-IIIA + prot. inhib. (at each time point) vs. MPS-IIIA (G). Scale bars : 10 μm (C, D, F).Source data are available online for this figure.

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