JAK2 (p-Y1007/Y1008) Rabbit pA

From 《Cancer Medicine》(2016.6): PublitionDirect effect of dasatinib on signal transduction pathways associated with a rapid mobilization of cytotoxic lymphocytes , IF:2.5
_x000D_ Author: Noriyoshi Iriyama, Yoshihiro Hatta & Masami Takei
_x000D_ Division of Hematology and Rheumatology, Department of Medicine, Nihon University School of Medicine, Tokyo, Japan

Blank control:HL-60
_x000D_ Primary Antibody (green line): Rabbit Anti-phospho-JAK2 (Tyr1007+Tyr1008) antibody (bs-2485R)
_x000D_ Dilution: 1μg /10^6 cells;
_x000D_ Isotype Control Antibody (orange line): Rabbit IgG .
_x000D_ Secondary Antibody : Goat anti-rabbit IgG-AF647
_x000D_ Dilution: 1μg /test.
_x000D_ Protocol
_x000D_ The cells were fixed with 4% PFA (10min at room temperature)and then permeabilized with 90% ice-cold methanol for 20 min at-20℃.The cells were then incubated in 5%BSA to block non-specific protein-protein interactions for 30 min at room temperature .Cells stained with Primary Antibody for 30 min at room temperature. The secondary antibody used for 40 min at room temperature. Acquisition of 20,000 events was performed._x000D_

P‌‌araformaldehyde-fixed, paraffin embedded (rat lung); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (phospho-JAK2 (Tyr1007+Tyr1008)) Polyclonal Antibody, Unconjugated (bs-2485R) at 1:2000 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

P‌‌araformaldehyde-fixed, paraffin embedded (rat spleen); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (phospho-JAK2 (Tyr1007+Tyr1008)) Polyclonal Antibody, Unconjugated (bs-2485R) at 1:2000 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining._x000D_ _x000D_

P‌‌araformaldehyde-fixed, paraffin embedded (mouse lung); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (phospho-JAK2 (Tyr1007+Tyr1008)) Polyclonal Antibody, Unconjugated (bs-2485R) at 1:2000 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining._x000D_ _x000D_

Tissue/cell: A549 cell; 4% P‌‌araformaldehyde-fixed; Triton X-100 at room temperature for 20 min; Blocking buffer (normal goat serum, C-0005) at 37°C for 20 min; Antibody incubation with (phospho-JAK2(Tyr1007+Tyr1008)) Polyclonal Antibody, Unconjugated (bs-2485R) 1:100, 90 minutes at 37°C; followed by a conjugated Goat Anti-Rabbit IgG antibody (bs-0295G-PE) at 37°C for 90 minutes, DAPI (5ug/ml, blue, C-0033) was used to stain the cell nuclei.

Tissue/cell: HeLa cell; 4% P‌‌araformaldehyde-fixed; Triton X-100 at room temperature for 20 min; Blocking buffer (normal goat serum, C-0005) at 37°C for 20 min; Antibody incubation with (phospho-JAK2(Tyr1007+Tyr1008)) Polyclonal Antibody, Unconjugated (bs-2485R) 1:100, 90 minutes at 37°C; followed by a conjugated Goat Anti-Rabbit IgG antibody (bs-0295G-AF488) at 37°C for 90 minutes, DAPI (5ug/ml, blue, C-0033) was used to stain the cell nuclei.

Tissue/cell: NIH/3T3 cell; 4% P‌‌araformaldehyde-fixed; Triton X-100 at room temperature for 20 min; Blocking buffer (normal goat serum, C-0005) at 37°C for 20 min; Antibody incubation with (phospho-JAK2(Tyr1007+Tyr1008)) Polyclonal Antibody, Unconjugated (bs-2485R) 1:100, 90 minutes at 37°C; followed by a conjugated Goat Anti-Rabbit IgG antibody (bs-0295G-PE) at 37°C for 90 minutes, DAPI (5ug/ml, blue, C-0033) was used to stain the cell nuclei.

Tissue/cell: A549 cell; 4% P‌‌araformaldehyde-fixed; Triton X-100 at room temperature for 20 min; Blocking buffer (normal goat serum, C-0005) at 37°C for 20 min; Antibody incubation with (phospho-JAK2(Tyr1007+Tyr1008)) Polyclonal Antibody, Unconjugated (bs-2485R) 1:100, 90 minutes at 37°C; followed by a conjugated Goat Anti-Rabbit IgG antibody (bs-0295G-AF488) at 37°C for 90 minutes, DAPI (5ug/ml, blue, C-0033) was used to stain the cell nuclei.

Blank control (Black line): Raji (Black).
_x000D_ Primary Antibody (green line): Rabbit Anti-phospho-JAK2(Tyr1007+Tyr1008) antibody (bs-2485R)
_x000D_ Dilution: 1μg /10^6 cells;
_x000D_ Isotype Control Antibody (orange line): Rabbit IgG .
_x000D_ Secondary Antibody (white blue line): Goat anti-rabbit IgG-PE
_x000D_ Dilution: 1μg /test.
_x000D_ Protocol
_x000D_ The cells were fixed with 4% PFA （10min）and then permeabilized with 90% ice-cold methanol for 20 min on ice. Cells stained with Primary Antibody for 30 min at room temperature. The cells were then incubated in 1 X PBS/2%BSA/10% goat serum to block non-specific protein-protein interactions followed by the antibody for 15 min at room temperature. The secondary antibody used for 40 min at room temperature. Acquisition of 20,000 events was performed._x000D_