Phospho-Syk (Y525 + Y526) Recombinant Rabbit Monoclonal Antibody [PSH03-70] - BSA and Azide free

☑ Cell treatment (CT)

Western blot analysis of Phospho-Syk (Y525 + Y526) on different lysates with Rabbit anti-Phospho-Syk (Y525 + Y526) antibody (HA750904) at 1/1,000 dilution and competitor's antibody at 1/1,000 dilution.

Lane 1: Ramos 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

Lysates/proteins at 20 µg/Lane.

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

Exposure time: Lane 1-2 (left): 1 minute 2 seconds; Lane 1-2 (right): 2 minutes 18 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 (HA750904) at 1/1,000 dilution and competitor's 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 serum starved for 16 hours add 12μg/mL human IgM (diluted in serum free medium) for 2 minutes labeling Phospho-Syk (Y525 + Y526) with Rabbit anti-Phospho-Syk (Y525 + Y526) antibody (HA750904) at 1/1,000 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 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 (Y525 + Y526) antibody (HA750904) 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 (M1305-2, 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.