Anti-Melanoma gp100 antibody [EP4863(2)] - Low endotoxin, Azide free

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Melanoma gp100 antibody [EP4863(2)] - Low endotoxin, Azide free (AB193201)

This data was developed using the same antibody clone in a different buffer formulation (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.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Melanoma gp100 antibody [EP4863(2)] - Low endotoxin, Azide free (AB193201)

This data was developed using the same antibody clone in a different buffer formulation (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.

• WB

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Western blot - Anti-Melanoma gp100 antibody [EP4863(2)] - Low endotoxin, Azide free (AB193201)

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab137078).

All lanes:

Western blot - Anti-Melanoma gp100 antibody [EP4863(2)] (<a href='/en-us/products/primary-antibodies/melanoma-gp100-antibody-ep48632-ab137078'>ab137078</a>) 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='/en-us/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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• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Melanoma gp100 antibody [EP4863(2)] - Low endotoxin, Azide free (AB193201)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab137078).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Melanoma gp100 antibody [EP4863(2)] - Low endotoxin, Azide free (AB193201)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab137078).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Melanoma gp100 antibody [EP4863(2)] - Low endotoxin, Azide free (AB193201)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab137078).

• IP

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Immunoprecipitation - Anti-Melanoma gp100 antibody [EP4863(2)] - Low endotoxin, Azide free (AB193201)

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab137078).

All lanes:

Immunoprecipitation - Anti-Melanoma gp100 antibody [EP4863(2)] (<a href='/en-us/products/primary-antibodies/melanoma-gp100-antibody-ep48632-ab137078'>ab137078</a>)

Predicted band size: 70 kDa

false

• WB

Unknown

Western blot - Anti-Melanoma gp100 antibody [EP4863(2)] - Low endotoxin, Azide free (AB193201)

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab137078).

All lanes:

Western blot - Anti-Melanoma gp100 antibody [EP4863(2)] (<a href='/en-us/products/primary-antibodies/melanoma-gp100-antibody-ep48632-ab137078'>ab137078</a>) 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