EGFR Recombinant Rabbit Monoclonal Antibody [SP00-86]

☑ Relative expression (RE)

Western blot analysis of EGFR on different lysates with Rabbit anti-EGFR antibody (ET1604-44) at 1/20,000 dilution and competitor's antibody at 1/2,000 dilution.

Lane 1: HeLa cell lysate
Lane 2: A431 cell lysate
Lane 3: MDA-MB-468 cell lysate
Lane 4: MCF7 cell lysate (low expression)

Lysates/proteins at 15 µg/Lane.

Predicted band size: 134 kDa
Observed band size: 175 kDa

Exposure time: 21 seconds; ECL: K1801;

4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1604-44) at 1/20,000 dilution and competitor's antibody at 1/2,000 dilution were used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

Immunocytochemistry analysis of A431 cells labeling EGFR with Rabbit anti-EGFR antibody (ET1604-44) at 1/500 dilution and competitor's antibody at 1/200 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-EGFR antibody (ET1604-44) at 1/500 dilution and competitor's antibody at 1/200 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.

☑ Knockout (KO)

All lanes: Western blot analysis of EGFR with anti-EGFR antibody [SP00-86] (ET1604-44) at 1:500 dilution.

Lane 1: Wild-type MC3T3 whole cell lysate.
Lane 2: EGFR knockout MC3T3 whole cell lysate.

Predicted band size: 134 kDa
Observed band size: 180 kDa

ET1604-44 was shown to specifically react with EGFR in wild-type MC3T3 cells. No band was observed when EGFR knockout samples were tested. Wild-type and EGFR knockout samples were subjected to SDS-PAGE. Proteins were transferred to a PVDF membrane and blocked with 5% NFDM in TBST for 1 hour at room temperature. The primary Anti-EGFR antibody (ET1604-44, 1/500) and Anti-Vinculin antibody (ET1705-94, 1/5,000) were used in 5% BSA at room temperature for 2 hours. Goat Anti-Rabbit IgG H&L (HRP) Secondary Antibody (HA1001) at 1:200,000 dilution was used for 1 hour at room temperature.

Cell lysate was provided by Ubigene Biosciences (Ubigene Biosciences Co., Ltd., Guangzhou, China).

Western blot analysis of EGFR on different lysates with Rabbit anti-EGFR antibody (ET1604-44) at 1/5,000 dilution.

Lane 1: Mouse placenta tissue lysate
Lane 2: Mouse lung tissue lysate
Lane 3: Rat skin tissue lysate
Lane 4: Rat liver tissue lysate

Lysates/proteins at 30 µg/Lane.

Predicted band size: 134 kDa
Observed band size: 175 kDa

Exposure time: 20 seconds; ECL: K1801;

4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1604-44) at 1/5,000 dilution was used in primary antibody dilution (K1803) at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

Flow cytometric analysis of A431 cells labeling EGFR.

Cells were fixed and permeabilized. Then stained with the primary antibody (ET1604-44, red) at 1/1,000 dilution and competitor's antibody (red) at 1/200 dilution, compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).

EGFR was immunoprecipitated from 0.2 mg HeLa cell lysate with ET1604-44 at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using ET1604-44 at 1/1,000 dilution. HRP Conjugated Anti-Rabbit IgG for IP Nano-secondary antibody at 1/5,000 dilution was used for 1 hour at room temperature.

Lane 1: HeLa cell lysate (input)
Lane 2: ET1604-44 IP in HeLa cell lysate
Lane 3: Rabbit IgG instead of ET1604-44 in HeLa cell lysate

Blocking/Dilution buffer: primary antibody dilution (K1803)
Exposure time: 30 seconds; ECL: K1801