Anti-MelanA antibody [EP1422Y]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-MelanA antibody [EP1422Y] (AB51061)

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.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-MelanA antibody [EP1422Y] (AB51061)

Intracellular 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).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MelanA antibody [EP1422Y] (AB51061)

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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MelanA antibody [EP1422Y] (AB51061)

Tissue Microarrays stained for Anti-MelanA antibody [EP1422Y] using ab51061 in immunohistochemical analysis. This table provides a detailed overview of positive (tick mark) and negative (cross mark) staining per sample type tested. The sections were pre-treated using Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0) for 20 minutes. The sections were incubated with ab51061 for 30 mins at room temperature followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

• WB

Lab

Western blot - Anti-MelanA antibody [EP1422Y] (AB51061)

Blocking buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-MelanA antibody [EP1422Y] (ab51061) at 1/20000 dilution

All lanes:

MeWo (Human malignant melanoma fibroblast) whole cell lysate at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 13 kDa

Observed band size: 20 kDa

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• WB

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Western blot - Anti-MelanA antibody [EP1422Y] (AB51061)

Blocking/Diluting buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-MelanA antibody [EP1422Y] (ab51061) at 1/20000 dilution

All lanes:

Human melanoma lysates at 15 µg

Secondary

All lanes:

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

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• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-MelanA antibody [EP1422Y] (AB51061)

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.

• OI-RD Scanning

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OI-RD Scanning - Anti-MelanA antibody [EP1422Y] (AB51061)

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.