Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human melanoma tissue sections labeling MelanA with purified ab51061 at 1/1000 dilution (1.15 µg/mL). Heat mediated antigen retrieval using Bond™™ Epitope Retrieval Solution 2 (pH 9.0). Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

Immunocytochemistry/ Immunofluorescence analysis of MeWo (Human malignant melanoma fibroblast) cells labeling MelanA with Purified ab51061 at 1/100 dilution (10 µg/mL). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1/200 (2.5 µg/mL). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1/1000 (2 µg/mL) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Anti-MelanA antibody [EP1422Y] (ab51061) at 1/20000 dilution (Purified) + Human melanoma lysates at 15 µg

Secondary
Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Predicted band size: 13 kDa
Observed band size: 20 kDa why is the actual band size different from the predicted?

Blocking/Diluting buffer: 5% NFDM/TBST

Flow Cytometry analysis of A375 (Human malignant melanoma epithelial cell, Left) / MeWo (Human malignant melanoma fibroblast, Right) cells labeling MelanA with Purified ab51061 at 1/1000 dilution (1 µg/mL) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

Overlay histogram showing Malme-3M cells stained with ab51061 (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 (ab51061, 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 image was generated using the un-purified format of the antibody.

Equilibrium disassociation constant (K_D)
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