Apologies if this is a repeat post. Our news server was being serviced when
last I tried posting...........
In response to my query about detection of fusion proteins using the 'Matchmaker' two-hybrid
system of Clontech I received the following informative replies that will
possibly interest other news-groupies. Many thanks to all those that responded
- I reckon the "Acknowledgments" page of my thesis will be crammed with
references to all the communicative, helpful and enthusiastic net users I have
had the luck to encounter. Ta!
1) I've just started getting into this twohybrid thang and whilst I havent
got too far yet, I may be able to offer a few pieces of information to
you! Im using the two hybrid matchmaker version II. The difference between
I and II is the strains and vectors involved, the upshot being that in MkII
you can make your life easier with a few more selection steps using
cycloheximide and you can use yeast mating to help isolate some of the
plasmids (saves lots of plasmid preps and transforming into Ecoli).
The other thing about the MkII version is that they claim it expresses the
fusion proteins at much higher levels than the original version, and that
it is now possible to detect the product by western blot. Whether this
means that you couldnt do this before I am not sure!
So far all I have done is clone my protein into the appropriate vector and
now I've just finshed checking the yeast phenotypes and the next step is
to start transforming a few plasmids into the yeast and see what happens
These are just the control experiments but hopefully I can get onto the
real thing in a week or so. It seem to be pretty labor unintensive as the
yeast take a few days to grow up, so Im only doing it part time right now.
The clontech kit seems well thought out, the manuals are pretty good, the
instructions are very comprehensive which is good for people like me who
have only experienced yeast via bread and beer.
I have a friend back home in the UK (yup, im a pom ;) who is much further
down the road with version I and he is going really well, no hassles what
soever... So, as far as I know, it seems like a good system.
Simon Twigger, Medical College of Wisconsin, Milwaukee.
2) We have used it very successfully with our own libraries. The level of
proteins transcribed from the truncated ADH promoter are very low. This
means nearly undetectable on Westerns (depending on your Ab).
Don't worry about it, we got positives although we never detected our
protein with an Ab good enough for immunoloc/whole mount. Look in
Development. September 95 Zachgo et al at the pictures to see how well
the Ab works and yet doesn't detect the fusion in the yeast.
>From the meeting on two Hybrid at the Max-Planck last Summer, the take
home lesson was, Clontech vectors are grat but stay away from their
libraries, they are rubbish.
Dr. Marcos Egea Cortines
MPI f|r Z|chtungsforschung
Kvln 50829 Germany
3) A few months ago I was using the Clonetech vectors and did not detect my
protein (as a fusion) by Western blot - I did not continue with the
experiment but plan to when time permits. I would appreciate hearing about
any replies you receive regarding your post. Thanks.
bschonho at magnus.acs.ohio-state.edu
4)I'm in the middle of doing the matchmaker thing now. I think I remember
that the book that comes with the kit says that they do. That of course is
a not great answer to your question.
However, as I am the lab molecular biologist/protein person and only do
westerns when I really need to, I'm waiting for our lab tech to get back
from vacation. When she returns (next week?), I will isolate some
extracts from yeasts containing plasmids with my coding region fused into
them and see if our Abs react on a blot.
John F. Hess, PhD
Email me jfhess at ucdavis.edu
5)I could find fusion proteins made by pAS2 easily, but you need fresh
cultures (that meens freshly transformed) and you should vortex the
yeast cells with glass beads and crack-buffer (Urea and SDS and Brom-
phenol blue. If you like, I can send you a protocol. Just give me your
adress and faxnumber.
Daniel Schlieper, dschliep at genetik.uni-koeln.de
6)I am using a different 2-H system, but I know from other people that they indeed
sometimes have problems to detect their expressed proteins on Western blots when
using the "Fields-vectors" of the Matchmaker, due to the crippled Adh-promoter.
Others, however, have successfully completed 2-H-screenings with this system.
One colleague from a neighbouring lab is just doing Western controls for her
constructs made with pGBT9 and I will forward your question to her. It is,
however, not only the vector which determines expression levels. I see different
levels for different fusions in the same vector (single copy but with full
length Adh promoter), so you might be fine with your construct in the same
vector that doesn't work for someone else with his constructs. Anyhow I would
strongly suggest not to embark for a screening when you do not see your hybrid
protein on Westerns, since I only get positive b-Gal stainings with the strongly expressing
constructs (when doing positive controls), and only very weak or no stainings
with the weak ones. Some people claim that the "Elledge-vectors", pAS and pACT,
show higher expression than pGBT9 / pGAD, although they also contain the short
Adh-promoter (check: Legrain et al., 1994, Nucl. Acids Res. 22, No.15, p. 3241).
I would just start with one vector and see what I get, the most important thing
probably is to have a copy of the Matchmaker manual, which is excellently
written and provides you with basically all information to be able to start
(vectors and strains for trying you can also get directly from the Fields- and
From: Andreas Kalmes, PhD.
e-mail: imsd018 at uni-wuerzburg.de
7) From the original kit, you cannot detect the fusion protein. Sorry.
However, there is a second (new and improved or so they say, and of course
it will cost more) kit that will be detectable by western, because it uses
different vectors. Check the 1996 cataloge.
Let me know if you need more help, and I will ask the master tech (or so I
call her) working with this system contact you.
Ben Lieberman, Ph.D.
.liebe003 at mc.duke.edu
8)In reply to your message I have tried the Clontech system for
screening known protein interactions but was rather frustrated with the kit
I got as proteins were expressed at very low amounts and were
subsequently undetectable on western blotting. This was a feature
built into the vectors to compensate for any toxicity the expressed
proteins may have on the yeast. However Clontech have sent me details
of their new Two-hybrid kit which is used in the same way as mine but the new
vectors allow increased expression and the subsequent detection
with a relative antibody. I think that they supply a MAB to the
relative activating and binding regions to aid detection. Also these
new vectors (pAS2-1 and pACT2) compare much more favourable in there
cloning sites when compared to pGBT9 and pGAD424 which I have and you
are not down to the level of messing around with T4 polymerase which
is a right bitch..
Anyway the kit is easy to use and I have had good results with it.