Anti-Phospho-EGFR (Tyr1068) Polyclonal Antibody is a Rabbit antibody targeting Phospho-EGFR (Tyr1068). Anti-Phospho-EGFR (Tyr1068) Polyclonal Antibody can be used in IHC-P,IHC-Fr,ICC/IF,IF,FCM.

IgG

Human,Rat (predicted:Mouse,Dog)

1. Paraformaldehyde-fixed, paraffin embedded (Rat skeletal muscle); 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 (p-EGFR (Tyr1068)) Polyclonal Antibody, Unconjugated (TMAB-10623) at 1:500 overnight at 4°C, followed by a conjugated secondary for 20 minutes and DAB staining.
2. Blank control (blue line): A431 (blue).
Primary Antibody (green line): Rabbit Anti-Phospho-EGFR (Tyr1068) antibody (TMAB-10623)
Dilution: 0.2 μg /10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG.
Secondary Antibody (white blue line): Goat anti-rabbit IgG-FITC
Dilution: 1 μg /test.
Protocol
The cells were fixed with 2% paraformaldehyde (10 min), then permeabilized with 90% ice-cold methanol for 30 min on ice. Cells stained with Primary Antibody for 30 min at room temperature. The cells were then incubated in 1 X PBS/2%BSA/10% goat serum to block non-specific protein-protein interactions followed by the antibody for 15 min at room temperature. The secondary antibody used for 40 min at room temperature. Acquisition of 20,000 events was performed.
3. Hela cell; 4% Paraformaldehyde-fixed; Triton X-100 at room temperature for 20 min; Blocking buffer (normal goat serum) at 37°C for 20 min; Antibody incubation with (phospho-EGFR (Tyr1068)) polyclonal Antibody, Unconjugated (TMAB-10623) 1:100, 90 minutes at 37°C; followed by a conjugated Goat Anti-Rabbit IgG antibody at 37°C for 90 minutes, DAPI (blue) was used to stain the cell nuclei.

IHC-P: 1:100-500; IHC-Fr: 1:100-500; ICC/IF: 1:100-500; IF: 1:100-500; FCM: 0.2μg/Test

Polyclonal

KLH conjugated Synthesised phosphopeptide: human EGFR around the phosphorylation site of Tyr1068

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.