WB
Western blot analysis of p38 alpha/MAPK14 on different lysates with Rabbit anti-p38 alpha/MAPK14 antibody at 1/1,000 dilution.
Lane 1: THP-1 cell lysate (10 µg/Lane),
Lane 2: C2C12 cell lysate (10 µg/Lane),
Lane 3: PC-12 cell lysate (10 µg/Lane),
Lane 4: Rat kidney tissue lysate (20 µg/Lane),
Lane 5: Mouse kidney tissue lysate (20 µg/Lane),
Exposure time: 3 minutes 10 seconds;
10% 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 1/1,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody at 1:100,000 dilution was used for 1 hour at room temperature.
ICC/IF
Immunocytochemistry analysis of RAW264.7 cells labeling p38 alpha/MAPK14 with Rabbit anti-p38 alpha/MAPK14 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 Rabbit anti-p38 alpha/MAPK14 antibody at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit 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-Mouse IgG H&L (594) was used as the secondary antibody at 1/1,000 dilution.
FC
Flow cytometric analysis of HeLa cells labeling p38 alpha/MAPK14.
Cells were fixed and permeabilized. Then stained with the primary antibody (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 at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).