M806

antibody from Invitrogen Antibodies

Targeting: EGF

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Other assay (Supportive data in Antibodypedia)

Antibody Data

Product number

M806

Provider

Invitrogen Antibodies

Product name

EGF Monoclonal Antibody (3A8)

Antibody type

Monoclonal

Antigen

Recombinant full-length protein

Description

The M806 anti-EGF antibody (Clone 3A8) has successfully been paired as the detection antibody in a sandwich ELISA with coating antibody M805 (Clone 1H11).

Antibody clone number

3A8

Concentration

1 mg/mL

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis of Human EGF was performed by loading 2 µg 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-mouse-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).

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescent analysis of EGF (green) in HeLa cells either left untreated (left panel) or treated with 10 ng/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.

Other assay

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

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 100 µL per well of Human EGF moncolonal antibody (Product # M805) in duplicate at 1, 3, 4, 5, 7, and 9 µg/mL in DPBS (Product # 28374) and incubating for 12-18 hours at 4C. The plate was aspirated and blocked with 300 µL per well of 4% BSA and 5% sucrose in DPBS for 1 hour at room temperature. Human EGF recombinant protein at 50 µL 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 50 µL of Human EGF biotinylated monoclonal antibody (Product # M806B) in all applicable wells at 0.2 µg/mL for 2 hours at room temperature. The plate was washed with ELISA Wash Buffer (Product # N503) and incubated with 100 µL 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 100 µL 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 450-550 nm