Anti-EGFR Polyclonal Antibody 4 is a Rabbit antibody targeting EGFR. Anti-EGFR Polyclonal Antibody 4 can be used in IHC-P,IHC-Fr,IF,FCM.

IgG

Human (predicted:Mouse,Rat,Rabbit)

1. Paraformaldehyde-fixed, paraffin embedded (Human brain glioma); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30 min; Antibody incubation with (EGFR) Polyclonal Antibody, Unconjugated (TMAB-05469) at 1:400 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructions and DAB staining.
2. Paraformaldehyde-fixed, paraffin embedded (Human lung carcinoma); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30 min; Antibody incubation with (EGFR) Polyclonal Antibody, Unconjugated (TMAB-05469) at 1:400 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructions and DAB staining.
3. Blank control: HUVEC cells (blue). Primary Antibody: Rabbit Anti-EGFR antibody (TMAB-05469), Dilution: 1 μg in 100 μL 1X PBS containing 0.5% BSA; Isotype Control Antibody: Rabbit IgG (orange), used under the same conditions); Secondary Antibody: Goat anti-rabbit IgG-PE (white blue), Dilution: 1: 200 in 1 X PBS containing 0.5% BSA.
Protocol
The cells were fixed with 2% paraformaldehyde (10 min), then permeabilized with 90% ice-cold methanol for 30 min on ice. Primary antibody (TMAB-05469,1 μg /1x10^6 cells) were incubated for 30 min on the ice, followed by 1 X PBS containing 0.5% BSA+10% goat serum (15 min) to block non-specific protein-protein interactions. Then the Goat Anti-rabbit IgG/PE antibody was added into the blocking buffer mentioned above to react with the primary antibody at 1/200 dilution for 30 min on ice. Acquisition of 20,000 events was performed.
4. Paraformaldehyde-fixed, paraffin embedded (Human brain glioma); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30 min; Antibody incubation with (EGFR) Polyclonal Antibody, Unconjugated (TMAB-05469) at 1:100 overnight at 4°C, followed by a conjugated Goat Anti-Rabbit IgG antibody for 90 minutes, and DAPI for nuclei staining.

IHC-P: 1:100-500; IHC-Fr: 1:100-500; IF: 1:100-500; FCM: 1μg/Test

Polyclonal

KLH conjugated synthetic peptide: human EGFR

Human

EGFR

Epidermal growth factor receptor

Receptor tyrosine kinase binding ligands of the EGF family and activating several signaling cascades to convert extracellular cues into appropriate cellular responses. Known ligands include EGF, TGFA/TGF-alpha, amphiregulin, epigen/EPGN, BTC/betacellulin, epiregulin/EREG and HBEGF/heparin-binding EGF. Ligand binding triggers receptor homo- and/or heterodimerization and autophosphorylation on key cytoplasmic residues. The phosphorylated receptor recruits adapter proteins like GRB2 which in turn activates complex downstream signaling cascades. Activates at least 4 major downstream signaling cascades including the RAS-RAF-MEK-ERK, PI3 kinase-AKT, PLCgamma-PKC and STATs modules. May also activate the NF-kappa-B signaling cascade. Also directly phosphorylates other proteins like RGS16, activating its GTPase activity and probably coupling the EGF receptor signaling to the G protein-coupled receptor signaling. Also phosphorylates MUC1 and increases its interaction with SRC and CTNNB1/beta-catenin.
Isoform 2 may act as an antagonist of EGF action.