[Yeast] Trouble detecting FLAG-tagged free ubiquitin via Western

Michael Dion via yeast%40net.bio.net (by MDion from CGR.Harvard.edu)
Tue Oct 9 15:26:39 EST 2007


Hi all,

Does anyone have any experience in detecting free FLAG-ubiquitin (~9.7
kDa) from yeast protein extracts?  I have built chromosomally integrated
genes of interest that express N-terminal FLAG-tagged ubiquitin DNA on
their 5' ends (i.e., FLAG-Ub-Gene).  Upon their translation, hydrolases
liberate the translated FLAG-Ub from the remainder of the translated
gene sequence.  I can detect these genes alone on Western blots (they
have a C-terminal myc tag), but cannot detect the liberated FLAG-Ub.

 

I have controls that strongly suggest the FLAG-Ub portions should be
present, and therefore detectable:  

1) The gene (with its myc tag) is detectable, and is of the correct
predicted size (i.e., its predicted size without FLAG-Ub).  By design,
if the gene is expressed, then FLAG-Ub, which is immediately N-terminal,
is expected to have been expressed.

2) I can detect FLAG in a construct in which liberation of the FLAG-Ub
sequence is prevented (by insertion of a proline residue immediately
following Ub).

3) I can detect free ubiquitin by anti-Ub Western; this cannot
distinguish between FLAG-Ub and Ub, but demonstrates that I can resolve
these small peptides.

 

Some possibilities:

a)       FLAG-Ub is unstable in yeast or in the protein extract.  We
don't favor this idea because FLAG-Ub is quite stable in a wide range of
other eukaryotic systems.  (It can also be purchased from Sigma!)

b)       Our clones aren't what we think they are.  We're sequencing
them, but our construction methodology makes this somewhat unlikely (the
parental strain with FLAG-Ub-<insertion point>-myc has been fully
sequenced).

 

Hoping you folks have some idea...we're reaching the end of our rope.

 


 

Michael Dion

Drummond Lab

FAS Center for Systems Biology

Harvard University

   

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