| Western blot (WB): | 1:500-2000 |
| Immunocytochemistry/Immunofluorescence (ICC/IF): | 1:50-400 |
| Flow Cytometry (Fixed): | 1:50-200 |

Western blot analysis of RAN using anti-RAN antibody (M00204-1). The sample well of each lane was loaded with 30 ug of sample under reducing conditions.
Lane 1: human 293T whole cell lysates,
Lane 2: human A375 whole cell lysates,
Lane 3: human Hela whole cell lysates,
Lane 4: human Jurkat whole cell lysates,
Lane 5: rat testis tissue lysates,
Lane 6: rat PC-12 whole cell lysates,
Lane 7: mouse testis tissue lysates.
After electrophoresis, proteins were transferred to a membrane. Then the membrane was incubated with mouse anti-RAN antigen affinity purified monoclonal antibody (M00204-1) at a dilution of 1:1000 and probed with a goat anti-mouse IgG-HRP secondary antibody (Catalog # BA1050). The signal is developed using ECL Plus Western Blotting Substrate (Catalog # AR1197). A specific band was detected for RAN at approximately 24 kDa. The expected band size for RAN is at 24 kDa.

ICC/IF analysis of Ran using anti- Ran antibody (M00204-1)
Ran was detected in immunocytochemical section of U20S cell. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (AR0022) for 15 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2μg/mL mouse anti- Ran Antibody (M00204-1) overnight at 4°C. Fluoro488 Conjugated Goat Anti-Mouse IgG (BA1126) was used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37°C. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

Flow Cytometry analysis of PC-3 cells using anti-RAN antibody (M00204-1).
Overlay histogram showing PC-3 cells stained with M00204-1 (Blue line). To facilitate intracellular staining, cells were fixed with 4% paraformaldehyde and permeabilized with permeabilization buffer. The cells were blocked with 10% normal goat serum. And then incubated with mouse anti-RAN Antibody (M00204-1) at 1:100 dilution for 30 min at 20°C. Fluoro488 conjugated goat anti-mouse IgG (BA1126) was used as secondary antibody at 1:100 dilution for 30 minutes at 20°C. Isotype control antibody (Green line) was mouse IgG at 1:100 dilution used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.

Western blot analysis of RAN using anti-RAN antibody (M00204-1). The sample well of each lane was loaded with 30 ug of sample under reducing conditions.
Lane 1: human 293T whole cell lysates,
Lane 2: human A375 whole cell lysates,
Lane 3: human Hela whole cell lysates,
Lane 4: human Jurkat whole cell lysates,
Lane 5: rat testis tissue lysates,
Lane 6: rat PC-12 whole cell lysates,
Lane 7: mouse testis tissue lysates.
After electrophoresis, proteins were transferred to a membrane. Then the membrane was incubated with mouse anti-RAN antigen affinity purified monoclonal antibody (M00204-1) at a dilution of 1:1000 and probed with a goat anti-mouse IgG-HRP secondary antibody (Catalog # BA1050). The signal is developed using ECL Plus Western Blotting Substrate (Catalog # AR1197). A specific band was detected for RAN at approximately 24 kDa. The expected band size for RAN is at 24 kDa.

ICC/IF analysis of Ran using anti- Ran antibody (M00204-1)
Ran was detected in immunocytochemical section of U20S cell. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (AR0022) for 15 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2μg/mL mouse anti- Ran Antibody (M00204-1) overnight at 4°C. Fluoro488 Conjugated Goat Anti-Mouse IgG (BA1126) was used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37°C. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

Flow Cytometry analysis of PC-3 cells using anti-RAN antibody (M00204-1).
Overlay histogram showing PC-3 cells stained with M00204-1 (Blue line). To facilitate intracellular staining, cells were fixed with 4% paraformaldehyde and permeabilized with permeabilization buffer. The cells were blocked with 10% normal goat serum. And then incubated with mouse anti-RAN Antibody (M00204-1) at 1:100 dilution for 30 min at 20°C. Fluoro488 conjugated goat anti-mouse IgG (BA1126) was used as secondary antibody at 1:100 dilution for 30 minutes at 20°C. Isotype control antibody (Green line) was mouse IgG at 1:100 dilution used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.


