Anti-Syk (phospho Y352) +ZAP70 (phospho Y319) 抗体 [EPR26232-39] (ab300398)

Blocking / Diluting buffer and concentration:5% NFDM/TBST

Exposure time: Lanes 1-4: 6 seconds; Lanes 5-6: 147 seconds.

Immunohistochemical analysis of paraffin-embedded mouse spleen without alkaline phosphatase treatment (Panel A), and mouse spleen with alkaline phosphatase treatment (Panel B), labelingSyk (phospho Y352) + ZAP70 (phospho Y319) with ab300398 at 1/500 dilution (0.944 µg/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on mouse spleen without alkaline phosphatase treatment (Panel A); no staining on mouse spleen with alkaline phosphatase treatment (Panel B). The section was incubated with ab300398 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

Immunohistochemical analysis of paraffin-embedded mouse lung without alkaline phosphatase treatment (Panel A), and mouse lung with alkaline phosphatase treatment (Panel B), labelingSyk (phospho Y352) + ZAP70 (phospho Y319) with ab300398 at 1/500 dilution (0.944 µg/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on mouse lung without alkaline phosphatase treatment (Panel A); no staining on mouse lung with alkaline phosphatase treatment (Panel B). The section was incubated with ab300398 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

Immunohistochemical analysis of paraffin-embedded mouse pancreatic cancer without alkaline phosphatase treatment (Panel A), and mouse pancreatic cancer with alkaline phosphatase treatment (Panel B), labelingSyk (phospho Y352) + ZAP70 (phospho Y319) with ab300398 at 1/500 dilution (0.944 µg/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on mouse pancreatic cancer without alkaline phosphatase treatment (Panel A); no staining on mouse pancreatic cancer with alkaline phosphatase treatment (Panel B). The section was incubated with ab300398 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

Immunofluorescent analysis of 4% Paraformaldehyde fixed, 0.1% Triton X-100 permeabilized Jurkat (human T cell leukemia T lymphocyte) labellingSyk (phospho Y352) + ZAP70 (phospho Y319) with ab300398 at 1/50 dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed secondary antibody at 1/1000 (2 μg/ml) dilution (green). Confocal image showing increased membranous staining in Jurkat cells treated with pervanadate (50 mM) for 5 min. The nuclear counter stain is DAPI (blue).

Tubulin is detected with ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) (red) at 1/200 dilution.

Immunofluorescent analysis of 4% Paraformaldehyde fixed, 0.1% Triton X-100 permeabilized EL4 (mouse lymphoma T lymphocytes) labelling Syk (phospho Y352) + ZAP70 (phospho Y319) with ab300398 at 1/50 dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed secondary antibody at 1/1000 (2 μg/ml) dilution (green). Confocal image showing increased membranous staining in EL4 cells treated with H2O2 (11 mM) for 10 min and then pervanadate (10 mM) for 30 min. The nuclear counter stain is DAPI (blue).

Tubulin is detected with ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) (red) at 1/200 dilution.

Flow Cytometry (Intracellular) analysis of Jurkat (human T cell leukemia cell line from peripheral blood) treated with 1mM pervanadate for 30 minutes (Red)/ Untreated control (Green) labeling Syk (phospho Y352) + ZAP70 (phospho Y319) with ab300398 at a 1/500 dilution. Cells were fixed with 4% paraformaldehyde and permeabilized with 90% methanol. A Goat Anti-Rabbit IgG (Alexa Fluor^® 488, (ab150081)) was used as the secondary antibody at a 1/2000 dilution. Black - Rabbit monoclonal IgG (ab172730) Isotype control. Blue - (unlabeled control) - Cells without incubation with primary and secondary antibodies. 

Flow Cytometry (Intracellular) analysis of EL4 (mouse lymphoma t lymphocyte) treated with 11mM H₂O₂ for 10 minutes then 10mM pervanadate for 30 minutes (Red)/ Untreated control (Green) labeling Syk (phospho Y352) + ZAP70 (phospho Y319) with ab300398 at a 1/50 dilution. Cells were fixed with 4% paraformaldehyde and permeabilized with 90% methanol. A Goat Anti-Rabbit IgG (Alexa Fluor^® 488, (ab150081)) was used as the secondary antibody at a 1/2000 dilution. Black - Rabbit monoclonal IgG (ab172730) Isotype control. Blue - (unlabeled control) - Cells without incubation with primary and secondary antibodies. 

Concentration of ab300398: 1/1000 dilution (0.472 μg/ml)

Secondary ab: Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051), 1/100000 dilution

Blocking/diluting buffer and concentration: 5% NFDM/TBST

Lane 1: Syk (phospho Y352) peptide a

Lane 2: Syk (phospho Y352) peptide b

Lane 3: Syk non-phospho peptide

Exposure time: 3 minutes