All lanes : Anti-Ubiquitin antibody [EPR8589] (ab137031) at 1/200 dilution

Lane 1 : MCF-7 Whole Cell Lysate
Lane 2 : MCF-7 Whole Cell Lysate + M132 (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. 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.

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 >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 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

All lanes : Anti-Ubiquitin antibody [EPR8589] (ab137031) at 1/1000 dilution

Lane 1 : Hela cell lysate
Lane 2 : Jurkat cell lysate
Lane 3 : 293T cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP conjugated Goat anti Rabbit IgG at 1/2000 dilution

Predicted band size: 8 kDa

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

Equilibrium disassociation constant (K_D)
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