SUMMARY Re: 50kD cross-reacting protein with 12CA5

[Namjin Chung]

The emails forwarded below are about the identity of a non-specific 50kD 
cytosolic protein which crossreact with 12CA5 anti-HA monoclonal antibody.

With help from many people, I feel I owe a summary of what I found about 
it to yeast community.
I found that many people have experienced the same problem as mine, and 
that have wondered what it could be.  Some people seem to know the answer 
while many others don't.

About the identity, Drs. Michael Snyder at Yale and Amit Kumar Srivastava 
at Carnegie-Mellon Univ suggested that it could be alcohol dehydrogenase 
(Adh1p).  The problem with this is that the calculated MW of Adh1p is 
only 37kD whereas the protein in question is apparently 50kD.  Srivastava 
mentioned that one possibility is glycosylation, and Adh1p does have four 
potential N-glycosylation sites.  However, I wonder whether four 
N-glycosylation could add up to 13kD, and whether N-glycosylation of 
cytosolic proteins is common.

There is another alcohol dehydrogenase protein, Adh4p, which could be 
about 50kD, but whether it is Adh4p is not confirmed.  The expression of 
the protein in question increases as cells undergo late-log phase or 
diauxic shift (M. Snyder and myself).  This is consistent with an idea 
that as cells grow in batch culture, they ferment initially and start to 
accumulate ethanol which could stimulate the expression of an alcohol 
dehydrogenase.

Dr. Lisa Frenz at MRC (UK) also had similar experience and she found that 
the non-specific band is strain-specific that she could detect it in 
cG378 strain, but not in W303.  I don't know if other netters had similar 
experience.  My strian had S288C background.

She also mentioned a reference (Gene 144 (1994) 63-68) which suggested to 
preincubate 12CA5 in crude yeast extract before use to avoid the 
non-specific band.  It could be a good idea theoretically, but I wonder 
if it will be convenient to do so.  Dr. Beatriz Satos at Yale provided 
with a company that sells another anti-HA monoclonal antibody which does 
not detect the 50kD non-specific band, but she said it now recognize a 
120kD non-specific protein, instead.  If one has a problem with 50kD 
non-specific like me, he/she can choose this antibody; vice versa.  FYI, 
this antibody is monoclonal clone 16B12, and the company and catalog no. 
are BAbCo (1-800-92-BABCO; www.babco.com) and MMS-101R).


PS: Many thanks to Drs. Michael Snyder, Beatriz Satos, Amit Kumar 
Srivastava, and Lisa Frenz for the helpful comments.


Namjin Chung
Dept of Pharmacology & Cancer Biology
Duke University Medical Center

Email: n.chung at duke.edu



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