Home >
Community >
Flag-tagged protein is not recognized
Upvote
24
Downvote
+ Proteins
+ Signaling
Posted by
Pablo Coronel
Flag-tagged protein is not recognized
Elena: The expected flag tag signal on your overexpressed protein may not be detectable due to many possible situations: 1. the tag is there but is hidden in the fused protein thus escaped detection; 2. the tag is not there due to degradation during biosynthesis. This can happen either at N- or C-termini. For (1), try to boil for 15 min in SDS/bMSH prior to loading to SDS-PAGE, and for (2) perform a peptide sequencing or MS detection for a purified fusion protein. Good luck.
Elena: The expected flag tag signal on your overexpressed protein may not be detectable due to many possible situations: 1. the tag is there but is hidden in the fused protein thus escaped detection; 2. the tag is not there due to degradation during biosynthesis. This can happen either at N- or C-termini. For (1), try to boil for 15 min in SDS/bMSH prior to loading to SDS-PAGE, and for (2) perform a peptide sequencing or MS detection for a purified fusion protein. Good luck.
I think Ke-Wei means beta-mercaptoethanol. I also think your protein is most likely truncated. This happens quite frequently. I assume you add protease inhibitor to your lysis buffer. If you do not, adding protease inhibitor during lysis might rescue your protein in case the degradation happens post-lysis. You can do a quick intracellular stain (fix cells in 3% formaldehyde for 15 min at RT, quench with 10 mM glycine for 3 x 5 min, permeabilize with PBS/0.5% BSA/0.5% saponin, and stain with anti-FLAG in the permeabilization buffer) and check by immunofluorescence or flow cytometry whether your tag is still intact as long as the protein is still inside the cell. If that intracellular stain turns out negative there is very likely a problem with the expression of your protein, in that it undergoes degradation/truncation immediately after synthesis. Perhaps you want to try moving the FLAG-tag to the other end then (from the N- to the C-terminus or the other way around).
I think Ke-Wei means beta-mercaptoethanol. I also think your protein is most likely truncated. This happens quite frequently. I assume you add protease inhibitor to your lysis buffer. If you do not, adding protease inhibitor during lysis might rescue your protein in case the degradation happens post-lysis. You can do a quick intracellular stain (fix cells in 3% formaldehyde for 15 min at RT, quench with 10 mM glycine for 3 x 5 min, permeabilize with PBS/0.5% BSA/0.5% saponin, and stain with anti-FLAG in the permeabilization buffer) and check by immunofluorescence or flow cytometry whether your tag is still intact as long as the protein is still inside the cell. If that intracellular stain turns out negative there is very likely a problem with the expression of your protein, in that it undergoes degradation/truncation immediately after synthesis. Perhaps you want to try moving the FLAG-tag to the other end then (from the N- to the C-terminus or the other way around).
Elena: The expected flag tag signal on your overexpressed protein may not be detectable due to many possible situations: 1. the tag is there but is hidden in the fused protein thus escaped detection; 2. the tag is not there due to degradation during biosynthesis. This can happen either at N- or C-termini.
For (1), try to boil for 15 min in SDS/bMSH prior to loading to SDS-PAGE, and for (2) perform a peptide sequencing or MS detection for a purified fusion protein. Good luck.
Elena: The expected flag tag signal on your overexpressed protein may not be detectable due to many possible situations: 1. the tag is there but is hidden in the fused protein thus escaped detection; 2. the tag is not there due to degradation during biosynthesis. This can happen either at N- or C-termini.
For (1), try to boil for 15 min in SDS/bMSH prior to loading to SDS-PAGE, and for (2) perform a peptide sequencing or MS detection for a purified fusion protein. Good luck.
More
VOTE
Thanks Ke-Wei for your reply. I'll try for sure to boil my samples more... but I've a question: what does bMSH stand for?
Thanks Ke-Wei for your reply. I'll try for sure to boil my samples more... but I've a question: what does bMSH stand for?
More
VOTE
I think Ke-Wei means beta-mercaptoethanol.
I also think your protein is most likely truncated. This happens quite frequently. I assume you add protease inhibitor to your lysis buffer. If you do not, adding protease inhibitor during lysis might rescue your protein in case the degradation happens post-lysis.
You can do a quick intracellular stain (fix cells in 3% formaldehyde for 15 min at RT, quench with 10 mM glycine for 3 x 5 min, permeabilize with PBS/0.5% BSA/0.5% saponin, and stain with anti-FLAG in the permeabilization buffer) and check by immunofluorescence or flow cytometry whether your tag is still intact as long as the protein is still inside the cell. If that intracellular stain turns out negative there is very likely a problem with the expression of your protein, in that it undergoes degradation/truncation immediately after synthesis. Perhaps you want to try moving the FLAG-tag to the other end then (from the N- to the C-terminus or the other way around).
I think Ke-Wei means beta-mercaptoethanol.
I also think your protein is most likely truncated. This happens quite frequently. I assume you add protease inhibitor to your lysis buffer. If you do not, adding protease inhibitor during lysis might rescue your protein in case the degradation happens post-lysis.
You can do a quick intracellular stain (fix cells in 3% formaldehyde for 15 min at RT, quench with 10 mM glycine for 3 x 5 min, permeabilize with PBS/0.5% BSA/0.5% saponin, and stain with anti-FLAG in the permeabilization buffer) and check by immunofluorescence or flow cytometry whether your tag is still intact as long as the protein is still inside the cell. If that intracellular stain turns out negative there is very likely a problem with the expression of your protein, in that it undergoes degradation/truncation immediately after synthesis. Perhaps you want to try moving the FLAG-tag to the other end then (from the N- to the C-terminus or the other way around).
More
VOTE