Phospho-Syk (Y352) Recombinant Rabbit Monoclonal Antibody [PSH11-66]

☑ Cell treatment (CT)

Western blot analysis of Phospho-Syk (Y352) on different lysates with Rabbit anti-Phospho-Syk (Y352) antibody (HA723362) at 1/5,000 dilution and pan Syk antibody at 1/1,000 dilution.

Lane 1: Ramos serum starved for 16 hours cell lysate
Lane 2: Ramos serum starved for 16 hours add 12μg/mL human IgM (diluted in serum free medium) for 2 minutes cell lysate
Lane 3: Ramos serum starved for 16 hours add 12μg/mL human IgM (diluted in serum free medium) for 2 minutes cell lysate, then the membrane treated with λpp for 1 hour

Lysates/proteins at 20 µg/Lane.

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

Exposure time: 30 seconds; ECL: K1801;
4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA723362) at 1/5,000 dilution and pan Syk antibody at 1/1,000 dilution were used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

☑ Cell treatment (CT)

Immunocytochemistry analysis of Ramos cells untreated / serum starved for 16 hours add 12μg/mL human IgM (diluted in serum free medium) for 2 minutes labeling Phospho-Syk (Y352) with Rabbit anti-Phospho-Syk (Y352) antibody (HA723362) at 1/1,000 dilution.

Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Phospho-Syk (Y352) antibody (HA723362) at 1/1,000 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (HA601187, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.

☑ Cell treatment (CT)

Flow cytometric analysis of Ramos cells untreated (left) / serum starved for 16 hours add 12μg/mL human IgM (diluted in serum free medium) for 2 minutes (right) labeling Phospho-Syk (Y352).

Cells were fixed and permeabilized. Then stained with the primary antibody (HA723362, 1/1,000) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).