WB
Western blot analysis of PI 3 Kinase p85 alpha on different lysates with Mouse anti-PI 3 Kinase p85 alpha antibody at 1/5,000 dilution.
Lane 1: SW480 cell lysate,
Lane 2: A431 cell lysate,
Lane 3: 293T cell lysate,
Lane 4: Jurkat cell lysate,
Lane 5: Raji cell lysate,
Lane 6: MCF7 cell lysate,
Lane 7: A549 cell lysate,
Lysates/proteins at 15 µg/Lane.
Exposure time: 1 minute 40 seconds;
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 at 1/5,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Mouse IgG - HRP Secondary Antibody at 1/50,000 dilution was used for 1 hour at room temperature.
ICC/IF
Immunocytochemistry analysis of U-937 cells labeling PI 3 Kinase p85 alpha with Mouse anti-PI 3 Kinase p85 alpha antibody 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 Mouse anti-PI 3 Kinase p85 alpha antibody at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Mouse IgG H&L (488) 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 (red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Rabbit IgG H&L (594) were used as the secondary antibody at 1/1,000 dilution.