1) It takes about 1-2 hours to pour the non-denaturing gel out and
prepare the samples, plus 2-4 hours to run the bandshift (=
electro-mobility shift assay, EMSA), the time will vary depending on the
size of the free probe, the concentration of the PAGE gel and the
maximum current you would choose to use. This will be followed by
1/2-12 hours to expose the film, if the probe is P-32 labeled. However,
the typical experiment will take longer if you plan to use different
probes - the labeled DNA that you would like to use is probably
double-stranded, and has to be less than 150 bp, better several dozen
base pairs long. The latter means you have to choose one of the
following: a) synthesize (partially) complementary oligos, anneal them,
fill them with Klenow and P-32 dNTP, run a gel and isolate them from a
gel; b) use "hot" PCR to amplify the probe either on a plasmid template
or on a longer synthetic oligo that would serve as a template; c)
prepare a restriction fragment from a cloned DNA, label it with Klenow
and isolate from a gel. Of course, if you a planning to use the same
type of DNA, you will have to prepare your probe only once or twice a
month (for P-32).
2) I would say that this is a qualitative, at best semi-quantitative
method.
3) The automation is up to you to develop. My guess is that for the
start it is better to pay somebody $30K to do it manually for a year (or
contract the job out to a university professor who can put three 10K
students on the project) than buy a robot for 1000K.
4) I would say the biggest limitations are caused by the
"non-physiological" type of the experiment and the "false positive"
results. Almost anything can bind (and shift) your probe in vitro in a
"pure probe - pure ligand" setting, but when you try a more stringent
method, such as a footprint, the results are often negative.
Competitive binding and the use of extracts/mixes instead of pure
ligands can improve the situation, but peer review process will nail you
down for using only bandshifts, they will probably ask you to add the
following additional experiments: a) supershift (antibody against the
ligand should supershift the band "ligand-DNA"); b) footprint (with
DNaseI); c) affinity column chromatography (your DNA probe attached to
the resin) followed by a sell gradient elution; d) if possible,
immunoprecipitation.
All of the above is for protein-DNA interaction. If your drug is a
small 300 Da molecule rather than a 50-200 KDa polypeptide, the
bandshifts may appear less convincing, given the fact that each
nucleotide in you probe is as big as your whole ligand. In addition,
you may have no antibodies against the ligand. At the other hand, your
purpose is not to publish in Cell, but to make money with the least
possible exposure to a peer-review process; therefore all the above
add-on experiments could be tossed aside (and again, with the risk of
running after something that does not exist in vivo for a year or two).
5) The results are reproducible.
Best regards,
victor
www.alphadna.com
-----Original Message-----
From: Association of Biomolecular Resource Facilities
[mailto:abrf-request@aecom.yu.edu]On Behalf Of mark
Sent: 18 octobre, 1999 19:17
To: Recipients of ABRF List
Subject: MolBio
Hello Everyone,
I am interested in using gel mobility shift electrophoresis to study
nucleic acid - drug interactions. Does anyone know the answers to any of
the following questions that I have?
In general ...
1) how long does it usually take to run one experiment?
2) how quantifyable are the results?
3) how automatable is the technique?
4) what are the limitations of the technique (what types of experiments
tend to fail)?
5) how reproducible are the results?
Thanks in advance for any replies to my questions. I think this
information will be useful to many people to have as a general review
and learning document, and I will post any "answers" sent to me in the
hopes that ABRF will add the final review document to its archives.
Dr. Mark McGovern
Vice President of Technology
SensorChem International Corp
Toronto, Canada