Immunohistochemical analysis of paraffin embedded Human colon tissue labelling Methionine Aminopeptidase 2/p67 with ab134124 at 1/250.

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

Overlay histogram showing HeLa cells stained with ab134124 (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 (ab134124, 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.

All lanes : Anti-Methionine Aminopeptidase 2/p67 antibody [EPR6887] (ab134124) at 1/1000 dilution

Lane 1 : HeLa cell lysate
Lane 2 : Caco 2 cell lysate
Lane 3 : HepG2 cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat anti-rabbit HRP conjugated antibody at 1/2000 dilution

Predicted band size: 52 kDa
Observed band size: 67 kDa why is the actual band size different from the predicted?

Equilibrium disassociation constant (K_D)
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