Anti-EGFR antibody (ab2430)

  Western blot - EGFR antibody (ab2430)

ab2430 at a 1/200 dilution staining A431 whole cell lysate. 

  Immunofluorescence - EGFR antibody (ab2430)

Cells were stained with anti-EGFR (ab2430; red) and DAPI (blue). A 1/40 dilution of ab2430 was used.

HeLa cells were fixed with 4% formaldehyde in PEM buffer. The coverslip was incubated in blocking buffer of 5% powdered milk in TBS-T plus 0.02% sodium azide for 1 hour at room temperature. Blocking buffer was removed and primary antibody was added at a dilution of 1/40 and incubated overnight at 4 degrees celsius. The coverslips were then washed 4-5 times with blocking buffer for 5 minutes. Secondary antibody was added at a dilution of 1/1000 and incubated at room temperature for one hour. From this point on coverslips were covered with foil to protect them from light. They were washed 5 times with TBS-T and then one time with PEM, for 5 minutes each wash. The coverslips were fixed 10-30 minutes in 4% formaldehyde in PEM buffer, then washed 3 times with PEM buffer for 5 minutes. 0.1M ammonium chloride in PEM buffer was added for 10 minutes to quench auto-florescence, and then slips were washed 2 times for 5 minutes in PEM followed by 3 washes for 5 minutes in TBS-T. Coverslips were then counterstained with DAPI in TBS-T for 1-2 minutes, TBS-T was then added and the coverslips mounted.

Michael Mancini, Ph.D.

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - EGFR antibody (ab2430)

ab2430 staining formalin fixed paraffin-embedded sections of human adhesion tissue.  The section was subjected to heat-mediated antigen retrieval in citrate buffer (pH 6.0) and blocked with 10% BSA for 1 hour prior to incubating with the primary antibody (diluted 1/200) for 12 hours at 4°C.  A biotinylated goat anti-rabbit antibody was used as the secondary.

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - EGFR antibody (ab2430)

ab2430 staining EGFR in human stomach tissue section by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections). Tissue underwent formaldehyde fixation before heat mediated antigen retrieval in 10 mM Citrate pH 6.0 and then blocking with 5% serum for 1 hour at 23°C was performed. The primary antibody was diluted 1/100 and incubated with sample for 1 hour at 23°C. A HRP conjugated goat polyclonal to rabbit IgG was used undiluted as secondary antibody.

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  Immunoprecipitation - EGFR antibody (ab2430)

ab2430 immunoprecipitate of EGFR in human AGS whole cell lysate. 100 µg of cell lysate was incubated with primary antibody (1/100 in RIPA buffer) and matrix (Protein A/G) for 16 hours at 4°C.
For western blotting Ab6721 (1/1000) was used.

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  Sandwich ELISA - EGFR antibody (ab2430)

Standard Curve for EGFR (Analyte: ab61264)dilution range 1pg/ml to 1ug/ml using Capture Antibody Mouse monoclonal [F4] to EGFR (ab62) at 5ug/ml and Detector Antibody Rabbit polyclonal to EGFR (ab2430) at 0.5ug/ml