Anti-SPHK1 antibody (ab71700) at 1/500 dilution + Lysate prepared from human MDA MB 231 cells at 15 µgSecondaryHRP-conjugated donkey polyclonal to rabbit IgG at 1/5000 dilutiondeveloped using the ECL techniquePerformed under reducing conditions.

Anti-SPHK1 antibody (ab71700) at 1/1000 dilution + mouse kidney tissue lysate at 12.5 µgSecondaryHRP goat anti-rabbit (H+L) at 1/5000 dilution

Immunohistochemical staining of SPHK1 in Human hepatocarcinoma tissue sections (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections) with ab71700 at a dilution of 1/25. Tissue was fixed with formaldehyde and blocked with 3% BSA for 0.5 hours at 38°C. Antigen retrieval was heat mediation with a citrate buffer (pH6). Samples were incubated with primary antibody (1/25) for 1 hours at 37°C. A HRP-conjugated goat anti-rabbit polyclonal (ready to use) was used as the secondary antibody.

ICC/IF image of ab71700 stained HeLa cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab71700, 5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.