General question about proteins tagged with small fusions: do you use a linker between your protein and a tag? What linker-how long and what sequence? Can the linker give me a problem? I added Flag to my retroviral vector (both N or C terminus) and I dont get expression of my fusion proteins. I sequenced the plasmid- all the sequences are correct and also my reading frame is correct.
I put Flag into BamHI site and AICDA (my gene) into EcoRI and XhoI of pMX retroviral vector. I added some nucleotides to create a linker between Flag and AICDA in hope that Flag would be separated by the linker and not obstructing my AICDA. However I have no protein expression (Flag western). gca ggt gga tcc gaa ttc sequence predicts a cryptic splice site. I tried to make cDNA from HEK cells transfected with these constructs with oligo dT primer and PCR for AICDA but got nothing.
Can you please give mew any advice?
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
Edited by KristinaZ, 10 October 2013 - 04:54 AM.