****************************************************************************** From: Gerhard Schwall Date: Wed, 13 Sep 2000 17:57:06 +0200 Subject: Silanizing eppendorf tubes? Organization: Fraunhofer IUCT Hi, in protein analysis it is common to silanize glass ware in order to prevent proteins from sticking to the vessel. My question is: Is it necessary to coat plastic vessels (e.g. polypropylene tubes like Eppendorf tubes) when dealing with low amounts of protein (for further MS analysis) and what reagents can be used. As far as I know Dichlorodimethylsilane and Chlorotrimethylsilane both require an -OH group at the tube surface to react. Any advise is appreciated. Thanks Gerhard e-mail: gschwall@iuct.fhg.de ****************************************************************************** From: "David Sparkman" Date: Wed, 13 Sep 2000 11:06:32 -0700 Subject: Re: Silanizing eppendorf tubes? Organization: CompuServe Interactive Services Gerhard, There are two different process, "silanization", a surface inactivation by means of a chemical reaction (usually with dimethyldichlorosilane -- DMCS) and "siliconization" surface deactivation by a process of coating the surface with a thin film of silicone oil (JT Watson, "Introduction to Mass Spectrometry", 3rd ED, footnote page 362. I am not sure which you mean, but I think you are talking about the latter. In either case, I don't think you will need to treat a plastic tube. I have no idea what DMCS would do to plastic tubing. Regards; O. David Sparkman Consultant-At-Large ods@compuserve.com ****************************************************************************** From: Rob Bossio Date: Wed, 13 Sep 2000 16:34:23 -0400 Subject: Re: Silanizing eppendorf tubes? Organization: Florida State University I would avoid using "silanized" eppendorfs, I used them once and got nothing but "silanzed" garbage in my ESI spectrum. Couldn't recognize anything, no protein, no nothing. I've never had a problem with using the polypropylene tubes, and I've even put some pretty nasty solvents in them (THF) with no ill effects. I would avoid them. Rob ****************************************************************************** From: "Daniel Boismenu" Date: Fri, 15 Sep 2000 18:22:16 GMT Subject: Re: Silanizing eppendorf tubes? Organization: * Greetings, The general course of action when working with trace level of proteins and peptides is to keep them in solution as much as possible. It is better to avoid putting them to dryness, if feasable. In some cases, you might not be able to redissolved lyophylized proteins or peptides efficiently. If you have problems redissolving a sample, use aqueous guanidine between 3 M and 6M. It is very useful and can be easily removed with a Zip Tip (Millipore). It is important to do the MS analysis as soon as the sample is ready. You should avoid letting the sample stand in an eppendorf tube with a solution of acetonitrile for too long. You might extract chemicals from the plastic that will interfere with the analysis. Concerning silanizing eppendorf tubes yourself, I would not recommend it. If you really need non-stick tubes, buy them. They are commercially available. Do not do it yourself, you only are going to mess up your lab with siloxanes. Once your lab is contaminated with siloxanes, it is very difficult to decontaminate it. Siloxane contamination is bad for trace level proteomics because the ions appear in the 500 m/z range, where doubly and triply charged peptides would appear. However, for some applications, commercial siliconized tubes might be the only alternative. A few months ago we had to sequence a synthetic peptide which was so hydrophobic that it would stick irreversibly to the eppendorf tube and the C18 Zip Tip. For that peptide, the solution was to ask the scientist to provide the sample in a siliconized tube and we did the cleaning with a C4 Zip Tip instead of the C18. If you have to use these tubes, you should check the amount of siloxane contamination in the ionization source and take the proper steps to decontaminate it, if necessary. Best Regards, Daniel Boismenu, Ph.D. Research Associate McGill University Mass Spectrometry Unit dboism1@po-box.mcgill.ca