Anti-Fyn antibody [EPR5500]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Fyn antibody [EPR5500] (AB125016)

Immunofluorescence staining of Neuro-2a (Mouse neuroblastoma cell line) cells with purified ab125016 at a working dilution of 1 in 500, counter-stained with DAPI. The secondary antibody was Alexa Fluor^® 488 goat anti rabbit (ab150077), used at a dilution of 1 in 500. ab7291 was used to stain tubulin, and this is shown in the top right hand panel. The cells were fixed in 4% PFA and permeabilized using 0.1% Triton X 100.

The negative control is shown in bottom middle and right hand panels - for the negative controls, purified ab125016 was used at a dilution of 1/200 followed by an Alexa Fluor^® 594 goat anti-mouse antibody at a dilution of 1/500.

• WB

Lab

Western blot - Anti-Fyn antibody [EPR5500] (AB125016)

False colour image of Western blot : Anti-Fyn antibody [EPR5500] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab125016 was shown to bind specifically to Fyn. A band was observed at 60 kDa in wild-type HEK-293 cell lysates with no signal observed at this size in FYN knockout cell line ab269630 (knockout cell lysate ab272440). To generate this image, wild-type and FYN knockout HEK-293 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 (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

All lanes:

Western blot - Anti-Fyn antibody [EPR5500] (ab125016) at 1/1000 dilution

Lane 1:

Wild-type HEK-293 cell lysate at 20 µg

Lane 2:

FYN knockout HEK-293 cell lysate at 20 µg

Lane 2:

Western blot - Human FYN knockout HEK-293 cell line (<a href='/en-us/products/cell-lines/human-fyn-knockout-hek-293-cell-line-ab269630'>ab269630</a>)

Predicted band size: 60 kDa

Observed band size: 60 kDa

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• WB

Lab

Western blot - Anti-Fyn antibody [EPR5500] (AB125016)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Fyn antibody [EPR5500] (ab125016) at 1/1000 dilution

Lane 1:

Jurkat (Human T cell leukemia cell line from peripheral blood) cell lysate at 20 µg

Lane 2:

Ramos (Human Burkitt's lymphoma cell line) cell lysate at 20 µg

Lane 3:

HEK-293 (Human epithelial cell line from embryonic kidney) cell lysate at 20 µg

Secondary

All lanes:

HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 60 kDa

Observed band size: 60 kDa

false

• WB

Unknown

Western blot - Anti-Fyn antibody [EPR5500] (AB125016)

All lanes:

Western blot - Anti-Fyn antibody [EPR5500] (ab125016) at 1/1000 dilution

Lane 1:

Ramos (Human Burkitt's lymphoma cell line) lysate at 10 µg

Lane 2:

Jurkat (Human T cell leukemia cell line from peripheral blood) lysate at 10 µg

Lane 3:

NIH/3T3 (Mouse embryo fibroblast cell line) lysate at 10 µg

Secondary

All lanes:

Goat anti-Rabbit HRP at 1/2000 dilution

Predicted band size: 60 kDa

Observed band size: 59 kDa

false

• WB

Lab

Western blot - Anti-Fyn antibody [EPR5500] (AB125016)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Fyn antibody [EPR5500] (ab125016) at 1/5000 dilution

Lane 1:

Mouse brain tissue lysate at 20 µg

Lane 2:

Rat brain tissue lysate at 20 µg

Secondary

All lanes:

HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 60 kDa

Observed band size: 60 kDa

false