We have constructed lentivirus transduced U251 cell lines to express (1) EGFRvIII (its amino acid sequence is MRPSGTAGAAFLALLAALCPASRALEEKKGNYVVTDHG..., which is identical to the sequence described in https://www.addgene.org/20737/)-mCherry or (2) EGFRvIII_G38C(MRPSGTAGAAFLALLAALCPASRALEEKKGNYVVTDHC...)-mCherry.
Both types of cells did not yield positive results by using antibodies against LEEKKGNYVVTDHC (https://www.novusbio.com/products/egfr-antibody-dh83_nbp2-50599af647).
And the (1) EGFRvIII-mCherry U251 cells were also tested negative using 2-step staining (https://www.emdmillipore.com/US/en/product/Anti-EGFRvIII-Antibody-clone-DH8.3,MM_NF-MABS1915-25UG + https://www.thermofisher.cn/cn/zh/antibody/product/Donkey-anti-Mouse-IgG-H-L-Highly-Cross-Adsorbed-Secondary-Antibody-Polyclonal/A32766),
The (2) EGFRvIII_G38C-U251 cells have not been tested by 2-step staining.
We are curious about this question. Have you met problems when dealing with EGFRvIII?
Antibodies against EGFRvIII, a variant of the epidermal growth factor receptor (EGFR) that is commonly found in certain types of cancer, may not work effectively for several reasons such as Tumor Heterogeneity, Immune Evasion Mechanisms, Resistance Mechanisms, Blood-Brain Barrier, Inadequate Targeting.
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