* Suggested working dilutions are given as a guide only. It is recommended that the user titrates the product for use in their own experiment using appropriate negative and positive controls.
Product Specific Information
The M806B anti-EGF antibody (biotinylated conjugate of clone 3A8) has successfully been paired as the detection antibody in a sandwich ELISA with coating antibody M805 (Clone 1H11).
Typical dilutions for sandwich ELISA: Coat = 1-3 µg/ml and Detection = 0.125-0.5 µg/ml.
The protein encoded by EGF gene is a growth factor that stimulates cell growth, proliferation, and differentiation by binding to its receptor EGFR. Human EGF is a 6045-Da protein with 53 amino acid residues and three intramolecular disulfide bonds
EGF Antibody (M806B) in WB M806_WB_Image_01162014.jpg
Western Blot with anti-EGF Monoclonal Antibody [3A8], Biotin conjugate (M806B)
Western blot analysis of Human EGF was performed by loading 2ug of recombinant human EGF (Lane 1) or supernatant from an EGF expression clone transfected in 293T cells onto a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked with 5% Milk/TBST for at least 1 hour. Membranes were probed with an EGF monoclonal antibody recognizing Human EGF (Product # M806) at a dilution of 1:5000 overnight at 4°C on a rocking platform. Membranes were washed in TBS-0.1%Tween 20 and probed with a goat anti-rabbit-HRP secondary antibody (Product # 31430) at a dilution of 1:10,000 for at least one hour. Membranes were washed and chemiluminescent detection performed using Super Signal West Dura (Product # 34075).
EGF Antibody (M806B) in IF EFG_Antibody_M806_Immunofluorescence_01162014.jpg
Immunofluorescence with anti-EGF Monoclonal Antibody [3A8], Biotin conjugate (M806B)
Immunofluorescent analysis of EGF (green) in HeLa cells either left untreated (left panel) or treated with 10ng/ml EGF (right panel) for 5 minutes. Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA (Product # 37525) for 15 minutes at room temperature. Cells were probed with an EGF monoclonal antibody (Product # M806) at a dilution of 1:50 for at least 1 hour at room temperature, washed with PBS, and incubated with DyLight 488 goat anti-mouse IgG secondary antibody (Product # 35502) at a dilution of 1:400 for 30 minutes at room temperature. F-Actin (red) was stained with DyLight 554 Phalloidin (Product # 21834) and nuclei (blue) were stained with Hoechst 33342 dye (Product # 62249). Images were taken on a Thermo Scientific ArrayScan at 20X magnification.
EGF Antibody (M806B) in ELISA M805 M806 M806B figure2.JPG
ELISA with anti-EGF Monoclonal Antibody [3A8], Biotin conjugate (M806B)
Sandwich ELISA analysis of Human EGF was performed using the Thermo Scientific ELISA Kit (Product # EHEGF) by coating a blank 96-well microtiter plate with 100µl per well of Human EGF moncolonal antibody (Product # M805) in duplicate at 1, 3, 4, 5, 7, and 9µg/ml in DPBS (Product # 28374) and incubating for 12-18 hours at 4C. The plate was aspirated and blocked with 300µl per well of 4% BSA and 5% sucrose in DPBS for 1 hour at room temperature. Human EGF recombinant protein at 50µl per well was added in duplicate at 500, 250, 125, 62.5, 31.25, 15.625, 7.8125 and 0 pg/ml for 2 hours at room temperature along with 50µl of Human EGF biotinylated monoclonal antibody (Product # M806B) in all applicable wells at 0.2µg/ml for 2 hours at room temperature. The plate was washed with ELISA Wash Buffer (Product # N503) and incubated with 100µl per well of Streptavidin-HRP (Product # N504) in all test wells at 1:130,000 dilution for 1 hour at room temperature and then washed and incubated with 100µl per well of TMB substrate (Product # 34028) for 30 minutes at room temperature in the dark. The plate was stopped with 0.16M sulfuric acid (Product # N600). Absorbances were read on a spectrophotometer at 450-550nm.
(This product is for In Vitro experimental use only. Not for resale without express authorization.)