EGF Antibody – Monoclonal

ELISA: This IgG fraction (at a concentration of 100 ng/ml) has been found by indirect ELISA to bind to hEGF at 1 ng/well and yield an OD greater than 1.0 under standard ELISA conditions

Immunocytochemistry: This monoclonal antibody has been found to stain specifically cell expressing human EGF at a concentration of 200 ng/ml. The ability of this monoclonal antibody to bind to hEGF was examined in cells fixed with neutral buffered formalin. The fixed cells were incubated for 20 min with 4% normal goat serum. reacted with 60 min with the diluted monoclonal antibody. and then with FITC-conjugated goat anti-mouse IgG. The immunofluorescent staining pattern was observed using epifluorescence microscopy

Western Blot: Western immunoblots resulted in a single band being detected at ~ 6.2 kDa at a concentration of 200 ng/ml

Western Blot Protocol: 1. After SDS-PAGE on 15% percent gels and electrophoretic transfer to PVDF membrane. block the membrane overnight with 4% normal goat serum in TBS/Tween-20 buffer as diluent.
2. Wash x 2 with TBS/Tween-20.
3. Apply the IgG fraction after diluting to a concentration of 200 ng/ml. (Note: more dilute solutions may be needed) Use 2% normal goat serum in TBS/Tween-20 as buffer for the primary antibody. 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