Leptin (LEP) Antibody – Monoclonal

Western Blot: Western immunoblots of adipocyte homogenates resulted in a single band being detected at ~ 16 kDa at a dilution of 1:4000.

Immunofluorescent: This ascites fluid has been found to stain specifically human adipocytes at a 1:1000 dilution. The ability of this monoclonal antibody to bind to leptin in adipocytes was examined in cells fixed with neutral buffered formalin. The fixed cells were incubated for 20 min with 4% normal goat serum. reacted for 60 minutes with the diluted mouse monoclonal antibody. and then with FITC-conjugated goat anti-mouse IgG. The immunofluorescent staining pattern was observed using epifluorescense microscopy.

Immunofluorescent: This ascites fluid has been found to stain specifically human adipocytes at a 1:1000 dilution. The ability of this monoclonal antibody to bind to leptin in adipocytes was examined in cells fixed with neutral buffered formalin. The fixed cells were incubated for 20 min with 4% normal goat serum. reacted for 60 minutes with the diluted mouse monoclonal antibody. and then with FITC-conjugated goat anti-mouse IgG. The immunofluorescent staining pattern was observed using epifluorescense microscopy.

Western Blot Protocol: 1. After SDS-PAGE on 12% percent gels and electrophoretic transfer to PVDF membrane. block the membrane overnight with 4% normal goat serum in TBS/Tween-20 buffer.
2. Wash x 2 with TBS/Tween-20.
3. Apply the ascites fluid after preparing a 1:4000 dilution. Use 2% normal goat serum in TBS/Tween-20. Let the primary antibody bind for 2-4 hours.
4. Wash x 3 with TBS/Tween-20.
5. Apply affinity purified HRP-goat anti-mouse IgG antiserum diluted 1:2500 (dilution may vary depending upon supplier) in 2% normal goat serum in TBS/Tween-20. Incubate 1-2 hours. Note: greater sensitivity may be achieved using ABC techniques.
6. Wash for 5 min x 4 in TBS/Tween-20.
7. Develop color using the enhanced DAB reaction.