Phospho-STAT5 (Y694) Recombinant Rabbit Monoclonal Antibody [SC05-31]

☑ Cell treatment (CT)

Western blot analysis of Phospho-STAT5 (Y694) on different lysates with Rabbit anti-Phospho-STAT5 (Y694) antibody (ET1610-48) at 1/1,000 dilution.

Lane 1: PC-12 cell lysate
Lane 2: PC-12 treated with 1mM Sodium orthovanadate for 30 minutes cell lysate
Lane 3: PC-12 treated with 1mM Sodium orthovanadate for 30 minutes cell lysate, then the membrane treated with λpp for 1 hour

Lysates/proteins at 20 µg/Lane.

Predicted band size: 91 kDa
Observed band size: 91 kDa

Exposure time: 3 minutes;
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 (ET1610-48) at 1/1,000 dilution was 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 PC-12 cells treated with 1mM Sodium orthovanadate for 30 minutes labeling Phospho-STAT5 (Y694) with Rabbit anti-Phospho-STAT5 (Y694) antibody (ET1610-48) at 1/100 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-STAT5 (Y694) antibody (ET1610-48) at 1/100 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.

☑ Cell treatment (CT)

Western blot analysis of Phospho-STAT5(Y694) on Hela cell lysates.

Lane 1: Hela cells, whole cell lysate, 10ug/lane
Lane 2/3: Hela cells treated with 100ng/mL IFN alpha for 60 minutes, whole cell lysates, 10ug/lane
Lane 4: Hela cells treated with 100ng/mL IFN alpha for 60 minutes, then treated with 2.8ug/ul lambda-PP for 30 minutes, whole cell lysates, 10ug/lane

All lanes :
Anti-Phospho-STAT5(Y694) antibody (ET1610-48) at 1:500 dilution. Anti-STAT5 antibody (ET1612-63) at 1:500 dilution. Anti-GAPDH antibody (ET1601-4) at 1:10,000 dilution. Goat Anti-Rabbit IgG H&L (HRP) (HA1001) at 1/200,000 dilution.

Predicted band size: 91 kDa
Observed band size: 91 kDa

Blocking and diluting buffer: 5% BSA.
Exposure time: 2 minutes 2 seconds

Immunohistochemical analysis of paraffin-embedded human breast cancer tissue with Rabbit anti-Phospho-STAT5 (Y694) antibody (ET1610-48) at 1/30,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET1610-48) at 1/30,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.