My target gene fusion with Flag , I detected target protein overexpressed in mammalian cells , but i can not detect Flag. The construct's sequence is correct withou frameshift.
What is the reason?
Where is your tag and which antibody you use?
Some FLAG antibodies will only recognize the tag if it is at the N-terminus of the protein, you can find this information on your antibody's datasheet.
Dear Kehua Jin
is not recognized in which approach? WB? ELISA? FACS?
Are you trasfecting mammalian cells?
In case you are performing FACS or ELISa, Are you sure that the domain that contain the tag is surface exposed?
best regards
Manuele
Kehua Jin, both these antibodies look like they should be able to recognize C-terminal FLAG.
1. Did you verify the sequence of your construct to check there is no problem that caused the FLAG to be out of frame?
2. Do you have an antibody for your protein that you can use to check for over-expression?
3. Does your lab has a plasmid that have worked for FLAG western in the past you can use as a positive control?
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