All lanes : Anti-RBX1 antibody [EPR6850(B)] (ab133565) at 1/1000 dilution

Lane 1 : MCF7 cell lysate
Lane 2 : HepG2 cell lysate
Lane 3 : HeLa cell lysate
Lane 4 : Jurkat cell lysate

Lysates/proteins at 10 µg per lane.

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

Predicted band size: 12 kDa

Immunohistochemical analysis of paraffin-embedded Human colon tissue labelling RBX1 with ab133565 at 1/100 dilution.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Overlay histogram showing HeLa cells stained with ab133565 (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 (ab133565, 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.

ab133565 at 1/70 immunoprecipitating RBX1 in HepG2 whole cell lysate.

For western blotting, a primary antibody was used at 1/1000 and a HRP-conjugated goat anti-rabbit IgG was used as the secondary antibody (1/1000).

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

Equilibrium disassociation constant (K_D)
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