(Apologize for longness!)

Dear Marie-Claude,

I am a little bit worried about your really interesting question, since you perhaps want to use a method for TEM preparations not many researchers have done in their career.

I know (I was student at that time = 70ies/80ies) to have seen the first "DNA-Protein-Spreittechnik"-results by KLEINSCHMIDT (e.g. for Hyaluronic acid backbone & glycosamino-proteoglycans) and also know the name of J. Dubochet from that time (who did NOTknow their contributions in TEM at that time ? [and today]).

Unfortunately I don't have the original copy of the DUBOCHET et al. 1971 article in my files (and it is not available online e.g. as PPV, so alternative "old methods" like "library lending" apply) but perhaps I shall get soon an electronic pdf of it by courtesy of the library of our University / Natural Sciences Faculty here in Salzburg . When I have this in hand, I shall send the copy for your personal use (only if you want).

I do not know anybody doing such DNA-preparations right now. (guessing that is a quite awsome procedure you have to follow) and have not seen specific literature on such methods used most recently..

But perhaps (hopefully) later on there will be a specific reply to you from a researcher who has found this your request.

I would like to point you meanwhile to

http://www.pnas.org/content/92/16/7535.full.pdf , where you can find at least a hint to "Protein-DNA complexes eluted in the excluded volume were deposited onto pentylamineactivated carbon-coated grids."

(I could tell you also some bibliographic citations additionally but don't like to "teach you" things you might have knowledge about already. I am quite sure about that you'll find no vendor of such special coated grids*), so you would have to make the pentylamine-coated (p.-activated) carbon-coated grids by yourself, using a kind of glow-charging apparatus (a technique which might be tricky for pentylamine or even Amylamine, and somehow hazardous task). Since for such reason (coating of carbon grids by charged ions for adhering DNA-Protein complexes) also (Williams RC, PNAS 74, pp2311ff, 1977) has been used I guess that all of those substances serve for (active/passive) "adhesion" of DNA molecules to the grid's (Formvar-)coated surfaces followed by staining e.g. with Uranyl acetate) or, as also Alcian Blue, for making the filmcoat on the grid surface more hydrophilic for your DNA-(-Protein)-load. With respect to this I most recently found also @

http://homepage.tudelft.nl/e3q6n/publications/2009/BJ2009_Faas_onlinesupp.pdf

or: http://www.biophysj.org/biophysj/supplemental/S0006-3495(09)01120-5

Supporting Material to Biophysical Journal, Volume 97

DNA deformations near charged surfaces: electron and atomic force microscopy views

D.I. Cherny, F.G.A. Faas, B. Rieger, L.J. van Vliet (2009)

(Article, free access in PMC as well as in CELL @

http://www.ncbi.nlm.nih.gov/pubmed/19686663 )

Biophys J. 2009 Aug 19;97(4):1148-57. doi: 10.1016/j.bpj.2009.06.015.

DNA deformations near charged surfaces: electron and atomic force microscopy views. by: Faas FG, Rieger B, van Vliet LJ, Cherny DI.

Source: Department of Imaging Science and Technology Delft University of Technology, Delft, The Netherlands.

Erratum in. Biophys J. 2010 Jun 2;98(11):2758

*) but it might perhaps be possible to use sophisticated "charged" silicon grids, e.g. from

I shall come back to this your intersting posting when I got the old article by Dubochet, Ducomunn, Zollinger and Kellenberger, 1971).

Best wishes & good luck meanwhile, regards, Wolfgang

PS: we can communicate also by Research Gate internal messaging if you like it that way. If you don't have (a) thought(s) about a suited glow-charging unit find some technical hints at:

http://www.yale.edu/pollard_lab/pdf/86.pdf

Dear Marie-Claude, I found one information lacking in my recent post (when finishing my post I overlooked that fact, so please apologize).

The lacking information (cf. *) above:

" *) but it might perhaps be possible to use sophisticated "charged" silicon grids, e.g. from ...." should have been:

"e.g. (hydroxyl) Smart Grids™ which are extremely hydrophilic or -perhaps better for your task: "Amine-terminated Smart Grids™" (from the catalogue: "In the life sciences, the amine terminal groups can be used to chemically attach a wide variety of biological molecules including proteins, antibodies, and nucleic acids through the use of bifunctional cross-linking agents such as imidoesters or N-hydroxysuccinimide-esters (NHS esters). Similar types of cross linking reagents can be used to covalently attach non-biological media such as nanoparticles functionalized with biomolecules. "

Cf. as an example: http://www.dunesciences.com/grids_functionalized.php#amine or http://www.2spi.com/catalog/grids/smartgrids/

(Disclaimer: no affiliation with one of the Companies mentioned or financial interest. There might be other vendors in addition...)

Best regards, Wolfgang

Thank you so much Wolgang for your very documented answer. Yes I would be very pleased if you can get a hand on the Dubochet paper (unfortunatly I can't find it even through our university library). Another way would be to directly request it from Dubochet himself (if he is still affiliated ?). The pentylamine protocole still seems to be used by some labs, so it may also be a way to get a protocol. From my inquiries, it seems that the old way using cytochromeC is even more tricky, that's why I am looking for another way to get nicely spread DNA. I have found a protocole with alcian blue, do you have any opinion on this ? I will have a look at the commercial reference either, all ways have to be investigated at this point!

Dear Marie-Claude,

meanwhile you should have gotten the publication you need.

Good to know that "Online" not always is sufficient for scientific work.

(also to my time of univ. studies) still always is fascinating since one can see what efforts have been done and results achieved in earlier () times of Electron Microscopy most people don't know of.

Adding only one information more: I've found a webpage for Prof. Dr. Jacques Dubochet, at least @ http://www.unil.ch/dee/page53292_en.html, where you also can find ( perhaps actual) private communication data.

Another source (publications) cf.:

http://www.labome.org/expert/switzerland/university/dubochet/jacques-dubochet-1295054.html

Best wishes, good luck and regards, Wolfgang

Dear Marie-Claude,

"pentylamine or iso-amylamine" technique developed by Dubochet for the protein-free spreading of DNA was substituted, because these amines are poisons, with two others: by Robley Williams, 1977 ("poly lysine technique", this is mentioned above) and by Keller (''BAC technique", Vollenweider HJ, Sogo JM, Koller T. A routine method for protein-free spreading of double- and single-stranded nucleic acid molecules. Proc Natl Acad Sci U S A. 1975 Jan;72(1):83-7.). Both last methods work well and reproducibly.

Good luck.

Dear Yuri, thank you for the references!

You are right that pentylamine has obviously been banned.

I have found some additional articles (1988, 1995,1999 as well as more general advice in M. A. Hayat 2000) dealing with glow discharge, adsorption methods/spreading using Alcian Blue and / or polylysine.

It seems to me that there are out several modifications how to proceed with the adsorption issue. In:

Electron Microscopy: Methods and Protocols [M. A. Nasser Hajibagheri (Ed.) ]

in METHODS in MOLEC. BIOL: Vol. 117, HUMANA PRESS Inc.Totowa, NJ. USA (1999), ISBN: 0-89603-640-5 (alk.paper)

(http://books.google.at/books?isbn=1592592015, search within the box for e.g.Cytochrome-c Spreading Procedure)

cf. Chapter 14 by: SCHNOS M & INMAN R.B.:

Applications of EM for Studying Protein-DNA Complexes

p229- (~239) Cytochrome-c Spreading Procedure, Direct Adsorption Method, etc.

perhaps for Marie-Claude: on p. 237: Reo-Virus core particles - Reo-RNA-species/units shown as spreaded threads.

HAYAT (in: Principles and Techniques of Electron Microscopy: Biological Applications (Chapter Support films), p.240/241 2000) tells:

(BAC = benzyldimethylalkylammoniumchloride (Lit.Ref. to Vollenweider et al, 1975).

So it may also depend on what the final task for the observations might be.

Regards, Wolfgang

(PS: Dear Marie-Claude if you would like to share the lit. collection I saved for my own interest, please just tell me...)

Thank you Wolfgang for the references. I should add that Koller's technique works with the protein-free spreading of RNA as well. For my knowledge, Jack Griffits, I think he is best in TEM of nucleic acids, preferred thin carbon films glow-discharged freshly to pick up DNA (RNA) from the monolayer. Actually TEM (and AFM!) of complexes of nucleic acids with proteins has a lot of tricks and rather close to the art than to the science.

Good afternoon,

Dear Yuri, again: Thank you... just to be sure about: with the (cf. your recent post) you meant the Article by Vollenweider et al, PNAS 1975 or is there another specific article by KOLLER Th. (?et al.?) I was not able to find...?

You certainly are right to point to Jack Griffith's (@Chapel Hill) experience with this matter.

For me it is really fascinating to see now how many papers and method approaches have been published meanwhile (late 1980ies/1990ies).

Best regards, Wolfgang

Thanks so much Wolfgang and Yuri for this very stimulating discussion. I am a bit nervous with the use of pentylamine, and seriously consider to test alcian blue or polylysine as we are starting from scratch anyway.

PS: Wolfgang, I did find most (if not all) of the litterature you mentioned, thanks for your concern

Dear Marie-Claude and Wolfgang, if you are interesting in the paper by Vollenweider, Sogo, Th. Koller, I can send PDF of it.

Dear Yuri, I was able to get the PNAS paper (if this is the one you're referring to). Thanks for your offer anyway.

Dear Yuri, apologize if I am late with my answer - this no attitude for being impolite - just having overlooked your friendly offer... No, thank you, I already had retrieved the article like Marie-Claude from PNAS.

Have a gorgious weekend,

best regards,

Wolfgang

Dear Marie-Claude. I was just reading the thread above and wondered how you ended up solving your problem. I've done a few different ways of spreading DNA onto grids but I do notice at times that the adhesion is not good. Could you share any successes you've had in techniques you've applied?

Dear Larry, no so far I did not give a try (not enough DNA to do the job). I could not think that this would be the bottleneck!

I will for sure let know all of you how things ended as soon as I will have give a try.