All lanes : Anti-Fyn antibody [EPR19636] (ab184276) at 1/1000 dilution

Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : FYN knockout HEK-293T cell lysate
Lane 3 : Human brain tissue lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 61 kDa
Observed band size: 159 kDa why is the actual band size different from the predicted?

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

Lane 1 : Human brain lysate
Lane 2 : Ramos (human Burkitt's lymphoma cell line) whole cell lysate
Lane 3 : HEK-293 (human epithelial cell line from embryonic kidney) whole cell lysate
Lane 4 : P0 mouse brain lysate
Lane 5 : P0 rat brain lysate

Lysates/proteins at 20 µg per lane.

Secondary
Lane 1 : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/4000 dilution
Lanes 2-5 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Developed using the ECL technique.

Predicted band size: 61 kDa
Observed band size: 60 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

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

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.

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.

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.

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.

All lanes : 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
Lane 2 : PC-12 (rat adrenal gland pheochromocytoma cell line) whole cell lysate
Lane 3 : NIH/3T3 (mouse embyro fibroblast cell line) whole cell lysate
Lane 4 : Rat spleen lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Developed using the ECL technique.

Predicted band size: 61 kDa
Observed band size: 60 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

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.

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.