Lane 1: Wild-type HAP1 cell lysate (40 µg)
Lane 2: SYK knockout HAP1 cell lysate (40 µg)
Lane 3: K562 cell lysate (40 µg)
Lane 4: Raji cell lysate (40 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab40781 observed at 75 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab40781 was shown to specifically react with Syk when Syk knockout samples were used. Wild-type and Syk knockout samples were subjected to SDS-PAGE. Ab40781 and ab8245 (loading control to GAPDH) were diluted at 1/5000 and 1/10000 dilution respectively and incubated overnight at 4C. 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 1/10000 dilution for 1 hour at room temperature before imaging.

Immunohistochemistry of paraffin embedded Human spleen tissue section labelling Syk with ab40781 at 1:5000 dilution (0.44 μg/ml).  Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). A ready to use ImmunoHistoProbe one step HRP Polymer (ready to use) was used as a secondary antibody at 1:0 dilution. PBS instead of primary antibody was used for negative control. Hematoxylin was used as a counterstain.

Immunocytochemistry/ Immunofluorescence analysis of Daudi (Human Burkitt's lymphoma lymphoblast) cells labeling Syk with Purified ab40781 at 1:150 (5 µg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1:200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1:1000 (2 µg/ml) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Flow Cytometry analysis of Raji (Human Burkitt's lymphoma B lymphocyte)  cells labeling Syk with Purified ab40781 at 1:70 dilution (10 μg/ml) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) secondary antibody was used at 1:2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

All lanes : Anti-Syk antibody [EP573Y] (ab40781) at 1/1000 dilution (Purified)

Lane 1 : Raji (Human Burkitt's lymphoma B lymphocyte) whole cell lysates
Lane 2 : Daudi (Human Burkitt's lymphoma lymphoblast) whole cell lysates
Lane 3 : WEHI-231 (Mouse B cell lymphoma B lymphocyte ) whole cell lysates

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 72 kDa
Observed band size: 72 kDa

Blocking/Diluting buffer: 5% NFDM/TBST

Anti-Syk antibody [EP573Y] (ab40781) at 1/1000 dilution (Purified) + Human bone marrow lysates at 15 µg

Secondary
Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Predicted band size: 72 kDa
Observed band size: 72 kDa

Blocking/Diluting buffer: 5% NFDM/TBST

Immunocytochemistry/Immunofluorescence analysis of Raji (human Burkitt's lymphoma B lymphocyte) labelling Syk with ab40781 at a dilution of 1:100 dilution (7µg/ml). Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Goat anti-rabbit IgG (Alexa Fluor^® 488, ab150077) (1:1000 dilution (2 µg/ml)) was used as the secondary antibody. The cells were co-stained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1:200 (2.5µg/ml). Nuclei counterstained with DAPI (blue). Control: PBS instead of the primary antibody.

Anti-Syk antibody [EP573Y] (ab40781) at 1/2000 dilution + Rat bone marrow lysate at 20 µg

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

Predicted band size: 72 kDa


Exposure time: 50 seconds

Blocking and diluting buffer: 5% NFDM/TBST. 

Anti-Syk antibody [EP573Y] (ab40781) at 1/5000 dilution + Mouse bone marrow lysate at 20 µg

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

Predicted band size: 72 kDa


Exposure time: 3 minutes

Blocking and diluting buffer: 5% NFDM/TBST.

Flow Cytometry analysis of K562 (human chronic myelogenous leukemia) cells labeling Syk with unpurified ab40781 at 1/70 (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) (1/2000 dilution) was used as the secondary antibody. Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) were used as the unlabeled control.