Anti-Ubiquitin antibody [EPR8589]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Ubiquitin antibody [EPR8589] (AB137031)

Immunofluorescence analysis of Jurkat cells labelling Ubiquitin with ab137031 at 1/250 dilution.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Ubiquitin antibody [EPR8589] (AB137031)

Overlay histogram showing HepG2 cells stained with ab137031 (red line). The cells were fixed with 4% paraformaldehyde (10 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 (ab137031 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 >5000 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 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

• WB

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Western blot - Anti-Ubiquitin antibody [EPR8589] (AB137031)

All lanes:

Western blot - Anti-Ubiquitin antibody [EPR8589] (ab137031) at 1/1000 dilution

Lane 1:

Hela cell lysate at 10 µg

Lane 2:

Jurkat cell lysate at 10 µg

Lane 3:

293T (Human embryonic kidney epithelial cell) cell lysate at 10 µg

Secondary

All lanes:

HRP conjugated Goat anti Rabbit IgG at 1/2000 dilution

Predicted band size: 26 kDa

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• WB

Lab

Western blot - Anti-Ubiquitin antibody [EPR8589] (AB137031)

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. ab137031 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 [EPR8589] (ab137031) at 1/200 dilution

Lane 1:

MCF-7 Whole Cell Lysate at 20 µg

Lane 2:

MCF-7 Whole Cell Lysate + M132 (50 uM 90 min) at 20 µg

Lane 3:

Mouse brain tissue lysate at 20 µg

Predicted band size: 26 kDa

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• OI-RD Scanning

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OI-RD Scanning - Anti-Ubiquitin antibody [EPR8589] (AB137031)

We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody. Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.