Anti-FGFR1 antibody [EPR806Y]

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-FGFR1 antibody [EPR806Y] (AB76464)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed 0.1% TritonX-100 permeabilized SH-SY5Y (Human neuroblastoma epithelial cell) cells labelling FGFR1 with at 1/100 dilution followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1/1000 dilution (Green). Confocal image showing cytoplasmic and weak nuclear staining in SH-SY5Y cell line is observed. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Used PBS instead of primary antibody secondary antibody is ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution.

• IP

Lab

Immunoprecipitation - Anti-FGFR1 antibody [EPR806Y] (AB76464)

ab76464 (purified) at 1/190 immunoprecipitating FGFR1 in 10 μg A-204 (Human muscle rhabdomyosarcoma)whole cell lysate (Lanes 1 and 2, observed at 145 kDa). Lane 3 - Rabbit monoclonal IgG (ab172730) instead of ab76464 in A-204 whole cell lysate. For western blotting, ab76464 at 1/500 and VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.

Blocking/Dilution buffer and concentration : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-FGFR1 antibody [EPR806Y] (ab76464)

Predicted band size: 92 kDa

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• WB

Lab

Western blot - Anti-FGFR1 antibody [EPR806Y] (AB76464)

Lane 1 : Wild type HAP1 whole cell lysate (20 μg)
Lane 2 : FGFR1 knockout HAP1 whole cell lysate (20 μg)
Lane 3 : MCF7 whole cell lysate (20 μg)
Lane 4 : SH-SY5Y whole cell lysate (20 μg)

Lanes 1 - 4 : Merged signal (red and green). Green - ab76464 observed at 140 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab76464 was shown to specifically react with FGFR1 when FGFR1 knockout samples were used. Wild-type and FGFR1 knockout samples were subjected to SDS-PAGE. ab76464 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1 μg/mL and 1/10000 dilution respectively. Blots were developed with 800CW Goat anti Rabbit and 680CW Goat anti Mouse secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-FGFR1 antibody [EPR806Y] (ab76464)

Predicted band size: 92 kDa

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• WB

Lab

Western blot - Anti-FGFR1 antibody [EPR806Y] (AB76464)

Western blot : Anti-FGFR1 antibody [EPR806Y] (ab76464) staining at 1/500 dilution, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab76464 was shown to bind specifically to FGFR1. A band was observed at 92 kDa in wild-type U-87 MG cell lysates with no signal observed at this size in FGFR1 knockout cell line. To generate this image, wild-type and FGFR1 knockout U-87 MG cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-FGFR1 antibody [EPR806Y] (ab76464) at 1/500 dilution

Lane 1:

Wild-type U-87 MG cell lysate at 20 µg

Lane 2:

FGFR1 knockout U-87 MG cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Observed band size: 92 kDa

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• WB

Lab

Western blot - Anti-FGFR1 antibody [EPR806Y] (AB76464)

Blocking/Dilution buffer and concentration : 5% NFDM/TBST.

All lanes:

Western blot - Anti-FGFR1 antibody [EPR806Y] (ab76464) at 1/500 dilution

All lanes:

SH-SY5Y (Human neuroblastoma cell line from bone marrow) cell lysate at 10 µg

Secondary

All lanes:

Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 92 kDa

Observed band size: 145 kDa

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