I am basically reposting this topic in a different section. Have you ever encountered this- I am having a problem to detect a fusion protein with the antibody to the tag. My protein is Flag-AICDA and I see the protein with anti-AICDA antibody but not with the Flag.
This is the sequence of the fusion junction:
ggatcc gcc acc ATG GAC TAC AAA GAC GAT GAC GAC AAG gga tca gca ggt gga tcc gaa ttc GAC AGC CTC TTG ATG AAC CGG
M D Y K D D D D K G S A G A S E F D S L L M N R
BamHI Kozac Flag BamHI EcoRI AICDA
My coleagues suggested that the fusion protein is translated from AICDA 6th aminoacid which is methionine.....but there is no Kozac sequence there-could this happen?
I need to use the tag for detection becuase I am going to study splice variants of this protein and the commercial antibodies are raised against the deleted regions.