Anti-Fyn 抗体 [EPR19636]

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-Fyn antibody [EPR19636] (AB184276)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed RAW 264.7 (mouse macrophage cell line transformed with Abelson murine leukemia virus) cell line labeling Fyn with ab184276 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) at 1/2000 dilution was used as the secondary antibody.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Fyn antibody [EPR19636] (AB184276)

Immunohistochemical analysis of paraffin-embedded human cerebral cortex tissue labeling Fyn with ab184276 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Cytoplasmic staining on human cerebral cortex (PMID : 7544314). Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Fyn antibody [EPR19636] (AB184276)

Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling Fyn with ab184276 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Cytoplasmic staining on T cells of human tonsil (PMID : 10523617). Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Fyn antibody [EPR19636] (AB184276)

Immunohistochemical analysis of paraffin-embedded rat testis tissue labeling Fyn with ab184276 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Membranous staining on rat testis (PMID : 7544314). Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Fyn antibody [EPR19636] (AB184276)

Immunohistochemical analysis of paraffin-embedded mouse spleen tissue labeling Fyn with ab184276 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Cytoplasmic staining on T cells of mouse spleen (PMID : 7544314, PMID : 10523617). Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IP

Supplier Data

Immunoprecipitation - Anti-Fyn antibody [EPR19636] (AB184276)

Fyn was immunoprecipitated from 0.35 mg of P0 mouse brain lysate with ab184276 at 1/40 dilution. Western blot was performed from the immunoprecipitate using ab184276 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1 : P0 mouse brain lysate 10 μg (Input).

Lane 2 : ab184276 IP in P0 mouse brain lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab184276 in P0 mouse brain lysate.

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

Exposure time : 3 minutes.

All lanes:

Immunoprecipitation - Anti-Fyn antibody [EPR19636] (ab184276)

Predicted band size: 60 kDa

false

• WB

Supplier Data

Western blot - Anti-Fyn antibody [EPR19636] (AB184276)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : Lanes 1 and 2 : 3 minutes; Lanes 3,4 and 5 : 8 seconds.

All lanes:

Western blot - Anti-Fyn antibody [EPR19636] (ab184276) at 1/1000 dilution

Lane 1:

Human brain lysate at 20 µg

Lane 2:

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

Lane 3:

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

Lane 4:

P0 mouse brain lysate at 20 µg

Lane 5:

P0 rat brain lysate at 20 µg

Secondary

Lane 1:

Western blot - VeriBlot for IP Detection Reagent (HRP) (<a href='/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/4000 dilution

Lanes 2 - 5:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 60 kDa

Observed band size: 60 kDa

true

• WB

Unknown

Western blot - Anti-Fyn antibody [EPR19636] (AB184276)

Lanes 1-3 : Merged signal (red and green). Green - ab184276 observed at 60 kDa. Red - loading control, ab8245 observed at 37 kDa.

ab184276 Anti-Fyn antibody [EPR19636] was shown to specifically react with Fyn in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266133 (knockout cell lysate ab257071) was used. Wild-type and Fyn knockout samples were subjected to SDS-PAGE. ab184276 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Fyn antibody [EPR19636] (ab184276) at 1/1000 dilution

Lane 1:

Wild-type HEK293T cell lysate at 20 µg

Lane 2:

FYN knockout HEK293T cell lysate at 20 µg

Lane 3:

Human brain tissue lysate at 20 µg

Predicted band size: 60 kDa

Observed band size: 60 kDa

false

• WB

Lab

Western blot - Anti-Fyn antibody [EPR19636] (AB184276)

False colour image of Western blot : Anti-Fyn antibody [EPR19636] 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, ab184276 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 [EPR19636] (ab184276) 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='/products/cell-lines/human-fyn-knockout-hek-293-cell-line-ab269630'>ab269630</a>)

Predicted band size: 60 kDa

Observed band size: 60 kDa

false

• WB

Supplier Data

Western blot - Anti-Fyn antibody [EPR19636] (AB184276)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Fyn antibody [EPR19636] (ab184276) at 1/1000 dilution

Lane 1:

RAW 264.7 (mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate at 10 µg

Lane 2:

PC-12 (rat adrenal gland pheochromocytoma cell line) whole cell lysate at 10 µg

Lane 3:

NIH/3T3 (mouse embyro fibroblast cell line) whole cell lysate at 10 µg

Lane 4:

Rat spleen lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 60 kDa,88 kDa

Observed band size: 60 kDa,90 kDa

true

Exposure time: 3min

• WB

Lab

Western blot - Anti-Fyn antibody [EPR19636] (AB184276)

Lanes 1-3 : Merged signal (red and green). Green - ab184276 observed at 60 kDa. Red - loading control, ab8245 observed at 37 kDa.

ab184276 Anti-Fyn antibody [EPR19636] was shown to specifically react with Fyn in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266133 (knockout cell lysate ab257071) was used. Wild-type and Fyn knockout samples were subjected to SDS-PAGE. ab184276 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Fyn antibody [EPR19636] (ab184276) at 1/1000 dilution

Lane 1:

Wild-type HEK-293T cell lysate at 20 µg

Lane 2:

FYN knockout HEK-293T cell lysate at 20 µg

Lane 2:

Western blot - Human FYN knockout HEK-293T cell line (<a href='/products/cell-lines/human-fyn-knockout-hek-293t-cell-line-ab266133'>ab266133</a>)

Lane 3:

Human brain tissue lysate at 20 µg

Predicted band size: 60 kDa

Observed band size: 159 kDa

false