Re: 2-D Gels & Imaging Systems (fwd)
Dirk S. Krapf (krapf@gilson.com)
Wed, 4 Feb 1998 08:57:18 -0800 (PST)
>---------- Forwarded message ----------
>Date: Fri, 23 Jan 1998 10:57:19 -0500
>From: Axel Ducret <axel_ducret@merck.com>
>To: Recipients of ABRF List <abrf@aecom.yu.edu>
>Subject: Re: 2-D Gels & Imaging Systems
>
>
>
>The only imaging 2D Software I know for MacIntosh is Melanie for the Power
>Mac.
>See their specs at the page http://expasy.hcuge.ch/melanie/melanie-top.html.
>However, that software (sold by the entremise of BioRad) is not cheap ...
>(around US$ 10K) but it is quite superior to other packages I know of.
>
>Axel Ducret
>
>P.S> My comments on ESA vs. Pharmacia were sent on October 23, 1997
>according to
>
>my mail software. It is enclosed in this mail as an attachment
>-------------------------------------------------------------------------------
>-
>
>
>Axel Ducret, Ph.D.
>Senior Research Biologist
>Merck-Frosst Canada Inc.
>Dept. Biochemistry and Molecular Biology
>P.O. Box 1005
>Pointe-Claire-Dorval PQ H9R 4P8
>Canada
>
>tel. + (514) 428-3428
>fax + (514) 428-4900
>
>
>JWLEONE@am.pnu.com wrote:
>
> Dear ABRFers,
>
> We are looking to purchase a 2D-gel electrophoresis system for the
> isolation of proteins amenable for mass analysis and/or protein
>sequencing.
> Emphasis will be on reliability of the system and reproducibilty of
> results.Some of the features we seek are: high throughput, automation
>and a
> good image analysis system. Any and all suggestions from all you 2D-gel
> people will greatly benefit us in making our decision.
>
> Regards,
>
> Joe Leone
>
> Joseph W Leone
> 7240-267-119
> Pharmacia & Upjohn
> 301 Henrietta St
> Kalamazoo, MI 49007 USA
> Tel: (616) 833-5375
> Fax: (616) 833-1488
> Email: joseph.w.leone@am.pnu.com
>
>
>Dear Joe,
>
>It just happens that I purchased a 2D-gel apparatus for my lab last
>month. My c
>riteria were the following: middle to high
>throughput (10 to 20 gels a week), high reproducibility, blotting of full
>gel, p
>ossibility of customization, high protein load.
>
>First, I considered the first (IEF) dimension possibilities, that is
>ampholine v
>s. immobilized pH gradient (IPG). Although
>ampholines-driven systems work well, they show some problems: difficulty
>to load
> large volumes or high concentration of
>proteins, pH drifts, difficulty to analyze very acidic or very basic
>proteins. T
>ube gels are also fairly tricky to manipulate (break
>easily). Therefore, I chose to go for the more general (and, in my view,
>more ro
>bust) method, that is IPG. Currently, the only
>manufacterer I would trust in this matter is Pharmacia (Multiphor II). ESA
>has (
>or used to have) an IPG tube gel system but it
>did not work very well (high M.W. proteins were excluded from the IEF
>step). Mor
>eover, Pharmacia sells pre-made IPG
>gradient and also the apparatus to cast your own ones. One multiphor
>apparatus a
>llows you to run 12 IEF simultaneously.
>Finally, Pharmacia also sell special reswelling unit in which you can load
>very
>high amounts of proteins directly in the gradient
>(they say in the mg range).
>
>Second, I considered the second dimension (SDS-PAGE). It should be able to
>run s
>imultaneously multiple gels (ideally, as
>many as you are running first dimensions), load the first dimension
>without pain
>ful modifications, be easy to manipulate
>(think, you are going to handle 10-20 gels at the same time, and they are
>rathe
>r big). The best fit I could find was a Hoefer
>IsoDalt 2nd dimension. One tank can run 10 gels simultaneously in a very
>homogen
>eous manner. Down points, though: thay
>are fairly expensive, you have to cast your own gels (they actually can
>also sel
>l you a system to automatize that step, but I
>would only consider this for a high throughput system), you cannot use a
>discont
>inuous buffer system (for SDS-PAGE, this is
>not an issue), and the IsoDalt is not meant as a stand-alone apparatus
>(i.e., yo
>u cannot do just straight SDS-PAGE because the
>gels lie on their side, preventing the load of liquid sample). The IsoDalt
>syste
>m is best used with a 20 gels capacity, that is 2
>tanks, because the caster will prepare 23 gels per load. If you are
>thinking to
>run only 10 gels at the time, then have a look at
>the ESA Investigator. Each tank can run 5 vertical gels simultaneously,
>and some
> gels can purchased pre-casted.
>
>Third, the blotting apparatus. Again, you need to have a system to blot
>all the
>gels you need simultaneously without to have to
>cut the gels first. Using the Hoefer apparatus, I decided to purchase the
>blotti
>ng kit that use the same tank as the second
>dimension (so there is no need to buy a separate equipment). For the ESA
>system,
> no luck: there is no commercially available
>system to blot the full gel (they are 20x25 cm), so you have to cut. You
>typical
>ly blot half of the gels you are running. So, for a
>20 gels system, the blotting kit of Hoefer allows me to blot 10 gels at
>the time
>. For the ESA system, for a 10 gels system, I
>would recommend 2 BioRad blotting apparatus (2x3 gels capacity).
>
>In summary that would be my choices:
>
>20 gels simultaneously: 2 Pharmacia Multiphor, 2 Hoefer Iso Dalt, 2 Hoefer
>blott
>ing kit.
>10 gels simultaneoulsly: 1 Pharmacia Multiphor, 2 ESA Investigator, 2
>BioRad blo
>tting kit
>
>Other considerations:
>
>staining gels: There is in my knowledge no system for staining these big
>gels si
>multaneously (especially if you have 20 of
>them). I am thinking to devise something for myself but I still do not
>know exac
>tly how this will look like. If you are using
>radioactivity, then it is not a problem.
>
>gel storage: The most convenient way to store gels is actually to keep
>them wet
>in 0.1% azide in a sealed bag ... except if you
>have enough gel dryers on hand.
>
>gel scanning: be ready to invest money in a good scanner (a dual
>phosphorimager/
>scanner is better) and a fast computer with a
>lot of RAM and harddisk. This will typically cost you as much as the
>2D-gel equi
>pment itself.
>
>imaging software: there are several softwares available, including Melanie
>II (
>http://expasy.hcuge.ch) and PDQUEST
>(http://www.pdiq.com) for the ones I know. Somebody could probably
>recommend oth
>ers. They will do about the same, but
>will run on different platforms (the preferred ones are still UNIX) and
>they hav
>e different choices of quantification. Somebody
>else could probably give you more detailed information about this.
>
>staffing: expect to have somebody working at least half time exclusively
>on the
>2D-PAGE system. This is a rather complicated
>process where it is easy to make mistakes. Do not expect to have good gels
>witho
>ut the proper staff. This is not something that
>should be runnig by Ph.D. students or an overworked technical staffed.
>This is t
>he best recipe for failure.
>
>Compatibility with MS and sequencing: same as with SDS-PAGE.
>
>Hope that will be of some help. Please contact me directly if you have
>some more
> questions.
>
>Axel Ducret
>
>
>
>Axel Ducret, Ph.D.
>Senior Research Biologist
>Merck-Frosst Canada Inc.
>Dept. Biochemistry and Molecular Biology
>P.O. Box 1005
>Pointe-Claire-Dorval PQ H9R 4P8
>Canada
>
>tel. + (514) 428-3428
>fax + (514) 695-0693