Human Total EGFR DuoSet IC ELISA

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-Store the unopened kit at 2-8 °C. -Bring all reagents to room temperature before use. -Reconstitute the Human Cytochrome c Standard with deionized or distilled water. Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making dilutions.	-When mixing or reconstituting protein solutions, always avoid foaming. -Substrate Solution should remain colorless until added to the plate. -Keep Substrate solution protected from light. -Stop Solution should be added to the plate in the same order as the Substrate Solution. -Wells that are green in color indicate that the Stop Solution has not mixed thoroughly with the Substrate Solution.

Upstream tips
-Store the unopened kit at 2-8 °C. -Bring all reagents to room temperature before use. -Reconstitute the Human Cytochrome c Standard with deionized or distilled water. Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making dilutions.
Protocol tips
-When mixing or reconstituting protein solutions, always avoid foaming. -Substrate Solution should remain colorless until added to the plate. -Keep Substrate solution protected from light. -Stop Solution should be added to the plate in the same order as the Substrate Solution. -Wells that are green in color indicate that the Stop Solution has not mixed thoroughly with the Substrate Solution.

Publication protocol

Assays to measure general Tyrosine-phosphorylation and total levels of endogenous EGFR in xenograft tissues were carried out according to the protocol described in the R&D Systems DuoSet IC Human PhosphoEGFR ELISA and DuoSet IC Human Total EGFR ELISA (R&D Systems, no. DYC1095 and DYC1854). Xenograft lysates were obtained and protein levels were quantified as described in the immunoblotting section. Nunc black MaxiSorp 96-well plates were coated with capture antibody and then blocked with SuperBlock buffer (ThermoFisher). Following the removal of the blocking solution, 50 µL of lysate (for pEGFR assay 3 µg of protein from H2073-SVD or 2 µg of protein from H2073-ASV; for tEGFR assay 0.75 µg of protein from H2073-SVD or 0.4 µg of protein from H2073-ASV) was transferred to the Nunc black MaxiSorp 96-well plates and incubated for 2 h. Following aspiration and washing of the plates with PBST, 50 µL of detection antibody was added and incubated for 2 h. Following aspiration and washing of the plates with PBST, 50 µL of Streptavidin-HRP was added to the tEGFR assay and incubated for 20 min in darkness. Following aspiration and washing of the plates with PBST. 50 µL of QuantaBlu fluorogenic peroxidase substrate (ThermoFisher) was added and incubated for 5 min. Fluorescence was read immediately afterwards on an Saffire plate reader using an excitation wavelength of 325 nm and an emission wavelength of 420 nm. Phospho-Tyrosine EGFR and total EGFR levels were calculated based on standard curve included in the assays. Phospho-Tyrosine EGFR levels were normalised to total EGFR levels and treatment groups were normalised to the mean of time-matched vehicle groups. Statistical significance was evaluated using a one-way, two-sided ANOVA test.

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Manufacturer protocol

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