The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 2 µg/ml. Predicted molecular weight: 37 kDa.
Use a concentration of 5 µg/ml. (paraformaldehyde fixed cells)
Western blot - Anti-AKR1C1/AKR1C2 antibody [4B6AF3] (ab131375)
All lanes : Anti-AKR1C1/AKR1C2 antibody [4B6AF3] (ab131375) at 2 µg/ml
Lane 1 : Molecular weight marker Lane 2 : HeLa Cell Lysate at 15 µg Lane 3 : HepG2 Cell Lysate at 15 µg Lane 4 : Human Liver Homogenate (HLH) at 10 µg Lane 5 : Human Heart Homogenate (HHH) at 10 µg Lane 6 : Mouse Liver Homogenate (MLH) at 10 µg Lane 7 : Rat Liver Homogenate (RLH) at 10 µg Lane 8 : Rat Heart Homogenate (RHH) at 10 µg
Secondary All lanes : Goat anti-Mouse AP at 1/3000 dilution
Predicted band size: 37 kDa
0.3% PBST was used as a blocking buffer in all incubation steps. The high background signal in Mouse tissue sample was caused by the direct reaction between the Mouse IgG in Mouse tissue preps and the Goat anti-Mouse secondary antibody.
Immunofluorescence analysis of AKR1C1 in HeLa cells, using ab131375 at 5 µg/ml. The cells were paraformaldehyde fixed (4%, 20 minutes) and Triton X-100 permeabilized (0.1%, 15 min). The cells were then incubated with ab131375 for 2h at room temperature or over night at 4°C. The secondary antibody was (red) Alexa Fluor® 594 Goat anti-Mouse IgG (H+L) used at a 1/1000 dilution for 1h. 1% BSA was used as the blocking agent for all blocking steps. The target protein locates to the cytosol.