Anti-MelanA antibody [EPR20380]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-MelanA antibody [EPR20380] (AB210546)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized MeWo (Human malignant melanoma cell line) cells labeling MelanA with ab210546 at 1/1000 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing cytoplasmic staining on MeWo cell line.

The nuclear counterstain is DAPI (blue).

Tubulin is detected with ab195889 (Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594)) at 1/200 dilution (red).

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) at 1/1000 dilution.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MelanA antibody [EPR20380] (AB210546)

Immunohistochemical analysis of paraffin-embedded human metastatic malignant melanoma tissue labeling MelanA with ab210546 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Cytoplasmic staining on human metastatic malignant melanoma is observed [PMID : 17445277] [PMID : 21840568].

Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MelanA antibody [EPR20380] (AB210546)

Immunohistochemical analysis of paraffin-embedded human malignant melanoma tissue labeling MelanA with ab210546 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Cytoplasmic staining on human malignant melanoma is observed [PMID : 17445277] [PMID : 21840568].

Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MelanA antibody [EPR20380] (AB210546)

Immunohistochemical analysis of paraffin-embedded human liver cancer tissue labeling MelanA with ab210546 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Negative control : no staining on human liver cancer [PMID : 10096358].

Counter stained with Hematoxylin.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-MelanA antibody [EPR20380] (AB210546)

Flow cytometry overlay histogram showing left Malme-3M positive cells and right negative HEK293 stained with ab210546 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilised with 0.1 % PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab210546) (1x 106 in 100μl at 0.2μg/ml (1/10500)) for 30min at 22°C.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min at 22°C

Isotype control antibody (black line) was Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

This antibody gave a positive signal in Malme-3M Fixed with 4% formaldehyde (10 min) / permeabilised with 0.1 % PBS-Triton X-100 for 15 min under the same conditions.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MelanA antibody [EPR20380] (AB210546)

Immunohistochemical analysis of paraffin-embedded mouse hair bulb tissue labeling MelanA with ab210546 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Cytoplasmic staining on melanocytes of mouse hair bulb is observed.

Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-MelanA antibody [EPR20380] (AB210546)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized B16F0 (Mouse melanoma cell line) cells labeling MelanA with ab210546 at 1/1000 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing cytoplasmic staining on B16F0 cell line.

The nuclear counterstain is DAPI (blue).

Tubulin is detected with ab195889 (Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594)) at 1/200 dilution (red).

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) at 1/1000 dilution.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-MelanA antibody [EPR20380] (AB210546)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed B16F0 (Mouse melanoma cell line) cells labeling MelanA with ab210546 at 1/500 dilution (red) compared with a rabbit monoclonal IgG isotype control (ab172730; black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (Alexa Fluor^® 488) at 1/2000 dilution was used as the secondary antibody.

• WB

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Western blot - Anti-MelanA antibody [EPR20380] (AB210546)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : Lane 1 and 2 : 10 seconds; Lane 3 and 4 : 3 seconds.

A-375 doesn't express MelanA. The expression profile observed is consistent with what has been described in the literature (PMID : 12670905).

All lanes:

Western blot - Anti-MelanA antibody [EPR20380] (ab210546) at 1/2000 dilution

Lane 1:

Human melanoma lysate at 20 µg

Lane 2:

A-375 (Human malignant melanoma cell line) whole cell lysate at 20 µg

Lane 3:

B16F0 (Mouse melanoma cell line) whole cell lysate at 20 µg

Lane 4:

MeWo (Human malignant melanoma cell line) whole cell lysate at 20 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 1/100000 dilution

Predicted band size: 13 kDa

Observed band size: 18 kDa

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