Anti-Eph receptor A2 antibody [EPR17660-120]

• Flow Cyt

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Flow Cytometry - Anti-Eph receptor A2 antibody [EPR17660-120] (AB185156)

Flow cytometric analysis of NIH/3T3 (mouse embryonic fibroblast cell line) cell line labeling Eph receptor A2 with ab185156 at 1/50 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/2000 dilution was used as the secondary antibody.

Total viable cells were gated for the FC image.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Eph receptor A2 antibody [EPR17660-120] (AB185156)

Immunofluorescent analysis of 100% methanol-fixed NIH/3T3 (mouse embryonic fibroblast cell line) cells labeling Eph receptor A2 with ab185156 at 1/50 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing membranous staining on NIH/3T3 cells.

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

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

• Flow Cyt

Lab

Flow Cytometry - Anti-Eph receptor A2 antibody [EPR17660-120] (AB185156)

Flow cytometry overlay histogram showing left NIH3T3 positive cells and right negative B16-F10 stained with ab185156 (red line). The cells were incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interactionfollowed by the antibody (ab185156) (1x 106 in 100μl at 5.0 μg/ml (1/424)) for 30min on ice.

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

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.

• IP

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Immunoprecipitation - Anti-Eph receptor A2 antibody [EPR17660-120] (AB185156)

Eph receptor A2 was immunoprecipitated from 0.35 mg of NIH/3T3 (mouse embryonic fibroblast cell line) lysate with ab185156 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab185156 at 1/500 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.

Lane 1 : NIH/3T3 whole cell lysate 10 μg (Input).

Lane 2 : ab185156 IP in NIH/3T3 whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab185156 in NIH/3T3 whole cell lysate.

Exposure time : 10 seconds.

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-Eph receptor A2 antibody [EPR17660-120] (ab185156)

Predicted band size: 108 kDa

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• WB

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Western blot - Anti-Eph receptor A2 antibody [EPR17660-120] (AB185156)

Exposure times Lane 1-3 : 3 seconds; Lane 4 : 3 minutes.

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Eph receptor A2 antibody [EPR17660-120] (ab185156) at 1/1000 dilution

Lane 1:

NIH/3T3 (mouse embryo fibroblast cell line) whole cell lysate at 20 µg

Lane 2:

L-929 (mouse connective tissue fibroblast cell line) whole cell lysate at 20 µg

Lane 3:

F9 (mouse embryonic testicular cancer cell line) whole cell lysate at 20 µg

Lane 4:

C6 (rat glial tumor 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 1/20000 dilution

Predicted band size: 108 kDa

Observed band size: 130 kDa

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