Anti-Melanoma gp100 antibody [EP4863(2)]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Melanoma gp100 antibody [EP4863(2)] (AB137078)

ab137078 staining Melanoma gp100 in human melanoma tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and antigen retrieval was by heat mediation in a EDTA buffer. Samples were incubated with primary antibody at a dilution of 1/250 for 30 minutes at room temperature. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody.

The immunostaining staining was performed on a Leica Biosystems BOND® RX instrument.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Melanoma gp100 antibody [EP4863(2)] (AB137078)

Flow cytometry overlay histogram showing left Malme-3M positive cells and right negative A-375 stained with ab137078 (red line). The cells were fixed with 4% formaldehyde (10 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 mins. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab137078) (1x 10⁶ in 100μl at 1.0μg/ml (1/500 dilution)) for 30 mins at 22°C.

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

Isotype control antibody (black line) was Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) 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.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Melanoma gp100 antibody [EP4863(2)] (AB137078)

Flow cytometry overlay histogram showing left B16-F10 positive cells and right negative Raw264.7 stained with ab137078 (red line). The cells were fixed with 80% methanol (5 mins) and then permeabilised with 0.1% PBS-Triton X-100 for 15 mins. The cells were then incubated in 1x PBS containing 10μg/ml anti CD16/CD32 and 10% normal goat serum to block FC receptors and non-specific protein-protein interaction followed by the antibody (ab137078) (1x 10⁶ in 100μl at 1.0μg/ml (1/500 dilution)) for 30 mins at 22°C.

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

Isotype control antibody (black line) was Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) 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 B16-F10 Fixed with 4% formaldehyde (10 min) / permeabilised with 0.1% PBS-Triton X-100 for 15 minss under the same conditions.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Melanoma gp100 antibody [EP4863(2)] (AB137078)

ab137078 staining Melanoma gp100 in B16-F0 (mouse skin) cells by ICC (Immunocytochemistry). Cells were fixed with 4% Paraformaldehyde and permeabilized with 0.1% Triton X-100. Samples were incubated with primary antibody at a dilution of 1/50. A goat anti rabbit IgG (Alexa Fluor® 488) (ab150077) was used as the secondary antibody at a dilution of 1/1000. Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) ab195889 was used as a counterstain at a dilution of 1/200. DAPI was used as a nuclear counterstain.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Melanoma gp100 antibody [EP4863(2)] (AB137078)

Overlay histogram showing B16-F0 (Mouse skin melanoma cell) cells stained with ab137078 (red line). The cells were fixed with 4% paraformaldehydeand then permeabilized with 90% methanol. The cells were then incubated with ab137078 at 1/40 dilution. The secondary antibody used wasGoat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution. Isotype control antibody (black line) was Rabbit monoclonal IgG (ab172730). Unlabelled control (blue line) was cells without incubation with primary antibody and secondary antibody.

• IP

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Immunoprecipitation - Anti-Melanoma gp100 antibody [EP4863(2)] (AB137078)

ab137078 immunoprecipitating Melanoma gp100. 10μg of cell lysate was incubated with primary antibody at a dilution of 1/500 and VeriBlot for IP Detection Reagent (HRP) (ab131366) at a dilution of 1/10000.

Lane 1 : B16-F0 (mouse melanoma epithelial cell-like) whole cell lysate 10ug
Lane 2 : B16-F0 (mouse melanoma epithelial cell-like) whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab137078 in B16-F0 whole cell lysate

Exposure time : 1 second

Observed MW : 85-100 KDa

All lanes:

Immunoprecipitation - Anti-Melanoma gp100 antibody [EP4863(2)] (ab137078)

Predicted band size: 70 kDa

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• WB

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Western blot - Anti-Melanoma gp100 antibody [EP4863(2)] (AB137078)

Exposure time : 18-0 seconds

Observed MW : 85-100 KDa

The molecular weights observed are consistent with what have been described in PMID : 16492709 and PMID : 7961886

All lanes:

Western blot - Anti-Melanoma gp100 antibody [EP4863(2)] (ab137078) at 1/1000 dilution

All lanes:

Human melanoma lysate 15μg

Secondary

All lanes:

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

Predicted band size: 70 kDa

false

• WB

Unknown

Western blot - Anti-Melanoma gp100 antibody [EP4863(2)] (AB137078)

Exposure time : 50 seconds

Observed MW : 85-100 KDa

The molecular weights observed are consistent with what have been described in PMID : 16492709 and PMID : 7961886

All lanes:

Western blot - Anti-Melanoma gp100 antibody [EP4863(2)] (ab137078) at 1/10000 dilution

All lanes:

B16-F0 (Mouse melanoma epithelial cell-like) whole cell lysate at 15 µg

Secondary

All lanes:

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

Predicted band size: 70 kDa

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