Phospho-AKT (T308) Recombinant Rabbit Monoclonal Antibody [PSH08-10]

☑ Cell treatment (CT)

Western blot analysis of Phospho-AKT (T308) on different lysates with Rabbit anti-Phospho-AKT (T308) antibody (HA722951) at 1/2,000 dilution.

Lane 1: Jurkat cell lysate
Lane 2: Jurkat treated with 100nM Calyculin A for 30 minutes cell lysate
Lane 3: HeLa cell lysate
Lane 4: HeLa treated with 100ng/mL Calyculin A for 30 minutes cell lysate
Lane 5: 293T cell lysate
Lane 6: 293T treated with 100nM Calyculin A for 15 minutes cell lysate

Lysates/proteins at 30 µg/Lane.

Predicted band size: 56 kDa
Observed band size: 56 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 (HA722951) at 1/2,000 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 Jurkat cells treated with or without 100nM Calyculin A for 30 minutes labeling Phospho-AKT (T308) with Rabbit anti-Phospho-AKT (T308) antibody (HA722951) 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-AKT (T308) antibody (HA722951) 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.

☑ Cell treatment (CT)

Flow cytometric analysis of Jurkat cells treated with or without 100nM Calyculin A for 30 minutes labeling Phospho-AKT (T308).

Cells were fixed and permeabilized. Then stained with the primary antibody (HA722951, 1μg/mL) (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).