Recombinant Anti-Syk antibody [EP573Y] (ab40781)

False colour image of Western blot: Anti-Syk antibody [EP573Y] 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, ab40781 was shown to bind specifically to Syk. A band was observed at 70/72 kDa in wild-type HEK-293T cell lysates with no signal observed at this size in SYK knockout cell line ab282649 (knockout cell lysate ab283048). To generate this image, wild-type and SYK knockout HEK-293T 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.

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.

Intracellular 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). 

Blocking/Diluting buffer: 5% NFDM/TBST

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.

Blocking and diluting buffer: 5% NFDM/TBST. 

Blocking and diluting buffer: 5% NFDM/TBST.

Intracellular 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.