Summary: Protein dimerisation in SDS gels
uj44228 at sunmailhost.lrz-muenchen.de
Tue Feb 13 12:08:52 EST 1996
In the end of January I have asked about dimerisation of proteins in SDS
gel. Here I want to give a short summary about the answers you gave me
during the last days.
First: YES several people have reported about proteins, which seems to
form dimeres or oligomers in SDS gels!
WHAT KINDS OF PROTEINS SHOW DIMER FORMATION IN SDS GELS?
I think, it is not easy to find out a common property.
- Most of the reported proteins have transmembrane domains, can bind to
membranes or fatty acids.
- Some of the reported proteins are relative small, although I was not
able to check it in each case (relative small means < 15-20 kd)
- There are examples of proteins which "are composed mainly of
beta-structure" and of proteins, where "no beta structures is present"
("... it's all-alfa helixes").
WHAT CAN BE THE REASONS FOR SDS / BETA-MERCAPTOETHANOS RESITENCE OF
CAN THE DIMER FORMATION IN SDS GELS BE SUPPRESSED?
No common reason for dimerisation in the presence of SDS is known.
Instead of this there are hints, which may indicate distinguishable
In the following I have made one speculative classification of reasons
with some reported tricks to suppress dimer formation. These tricks may
work or not - depending on the nature of each protein.
1) the proteins are not denaturated well enough and can still
This may be supported by the fact that proteins can dimerise although
they do not contain cysteins.
The partial denaturation may be especially the case for smaller
proteins. Long time ago I have read that e.g. in the average one
SDS-molecules binds per about 40 amino acids (is that right?).
-> Use SDS gels with aditional 6 M urea.
-> Boil your samples. Some people said, that their dimers are resitant
to SDS and 8 M urea, but comes apart when boiled.
2) not all intermolecular disulfide bonds are broken
Yes, disulfide bonds, that is what we learn in the school ;-)
The dimerisation of several proteins was reported to be depending on the
presence of cystein.
-> Pretreatment with b- mercaptoethanol normaly suppressed the
-> Mix also an amount of b-ME to the gel
-> Use 25 mM DTT instead of b-ME
-> Again: Some people said, that their dimers are resitant to SDS but
comes apart when boiled. (Did they mean boiling with b-ME or DTT?).
Some people mentioned, that they have seen oligomers only with purified
proteins. Can this indicate, that otherwise the proteins react with
other proteins in an unspecific way (->smear?), so that no clear
oligobands can be seen?
-> do not purify your protein, hahahahaa!
4) other possibilities
There are other possibilites for dimerisation of proteins. Lauge thinks
"that dimers in the 0.01-0.1% range can be explained by natural chemical
crosslinking mechanisms such as transamidations".
It was also speculated that dimerisation has something to do with
glycosylation AND high hydrophobicity of some proteins.
The following tricks *can* suppress dimerformation in gels. Maybe they
have something to do with transamidations or the dependency of
-> Do NOT heat the protein samples in Laemmli-buffer. Instead of the
incubate 5 minutes at 37 degree C and minimise the time from sample
dissolution in SDS loading buffer. (I have done it. Yeah, it reduces my
dimer bands! Lutz)
-> Check: Boil your sample for a longer time and look, if you will get
stronger dimer bands. This may give you a hint about your dimers.
-> Ad some sucrose to the loading buffer
ARE THERE ANY PUBLISHED COMMENTS ABOUT SDS-RESITENCE OF DIMER
Look yourself to the following references. I thing some of them are
helpfull for further answers (and questions?).
Biochemistry 1992, 31, 12719-25
Journal of Biological Chemistry vol 267, No 11, Apr 15 pp. 7683-89
Tokunaga, M et al. (1979) European J. Biochem. 95: 441-448
Yu, F et al. (1979) FEBS Letters 100: 71-74
Griffin, T. J. and Kolodner, R. D., Purification and preliminary
characterization of the Escherichia coli K-12 RecF protein, Journal of
Bacteriology, 172, 6291, 1990.
PNAS 90: 3840-3844
MANY THANKS TO ALL WHO HAVE PARTICIPATED TO THIS DISCUSSION:
Anthony A. Bominaar
Alberto I. Roca
Lutz Essers, Universitaet Munich
E-Mail: uj44228 at sunmail.lrz-muenchen.de
(NEW E-Mail, starting within the next days:
Essers at gen1.genetik.uni-koeln.de)
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