Len
Thank you for the posting. It is always entertaining to have a major
marketing company admit some limits. Do you think that they will now
come out with a product for crow, having acquired a good taste of it
here?
Dr. Alpert and I have posted several times on the use of HILIC for
detergent removal, as well as for phosphopeptide retention, rather
than the IMAC approach. However, there has not been much feedback on
this subject. I am curious as to whether anyone has had success with
this? (One underutilized function of this discussion group would be
to allow more manufacturer postings so that the free market of ideas
would more quickly move us onto better products or techniques. We
have to agree to be civil about it though!)
Part of the problem with the HILIC approach is that the salt
concentrations are somewhat high in in-gel digests and one needs to
carefully manage this aspect of the problem so that the polar
peptides will actually stick. Keeping the molarity below 50mM is
only a benchmark level. It may require even less salt present, so a
C18 step is not out of the question here, but the salt tolerance of
HILIC methods needs to be better defined.
Additionally the capacity of ZipTips or their competitors (PrepTips
and SuproTips) are somewhat low as compared to MiniSpin columns with
HILIC chemistries. The liquid volumes of spin columns are not
substantially larger than the coated wall or plugged flow tips, but
the bed volumes are much greater. This means that the amount of polar
solute retained could be greater, and you would not need as clean a
sample to be successful at this.
We have samples of both types of products available for those of you
interested in framing this problem in more realistic terms other than
the standards that have been run. Please let me know if you are
interested.
I hope to see many of you at San Diego where we can discuss this further.
Sincerely,
Amos Heckendorf (amos@nestgrp.com)
508.481.6223
----------
>I am forwarding some feedback from Millipore, vendors of ZipTip, on
>the recent IMAC ZipTip discussion as I think our members would be
>interested to read them.
>
>
>
>Hi Len:
>
> I've been watching your messages on the newsgroup and have a few
> comments. I am the inventor of ZipTip and am responsible for new product
> development. The introduction of ZipTipMC for phosphopeptides was a
> balancing act. After reading the literature on the use of IMAC for
> phosphopeptides, it was clear that scientists were quite split on
> whether it really worked. However, for those who managed to get the
> chemistry to work, it was quite beneficial, because there was no other
> easy way. When we launched the product, we knew that it was not going to
> work for all sequences and hence we put the disclaimers in the operating
> instructions. Nonetheless, we felt that the product could provide a
> useful tool to some customers. This was a risky decision because it
> balanced the possibility of helping a portion of our customers while
> potentially upsetting others- - -possibly to the detriment of the ZipTip
> reputation.
>
> Looking back, if I had to do it all over, I would probably still [have]
> launched it. The reason is that we have helped a few labs to advance
> their research. Fundamentally, having IMAC in a ZipTip is a good idea,
> if just to have the chemistry capability. However, from a product
> introduction standpoint, it really helps to have an application that
> scientists can relate to, hence phosphopeptides.
>
> The issue that we are encountering is that there are others like
> yourself that are not having good luck with the IMAC chemistry in this
> application. For this, I would like to personally apologize for any
> inconvenience that this has caused. I hope that the explanation above
> explains our reasoning.
>
>Millipore
>
>
>
>You need not offer apologies. I was one of the researchers pressing
>Millipore to make IMAC ZipTips as it is a potentially useful tool for
>those of us who do not have the benefits of triple quads. However, as
>you say it is not an easy science and for some is already proving
>its worth. Are you continuing to investigate this product in terms of
>exploring different chemistries or applicability to different types
>of phosphorylated sequences (rather than just beta-casein)?
>
>Len
>
>
>Regarding additional investigation of the IMAC chemistry, with the
>exception of a few low key outside collaborations, we've probably closed
>the book on this chapter. Although the subtleties of sequence/PO4 as it
>relates to IMAC binding would be a fascinating graduate research project,
>unfortunately its not something that I can pursue at this time.
>
>Lastly, if you are going to ABRF (I won't be attending), I would like to
>invite you to view our poster on hydrophilic interaction chromatography.
>ZipTips with this chemistry were shown to very effective for removing
>detergents and stains from peptides.
>
>Millipore
>
>
>--
>*********************************************************************
>Dr Len C. Packman
>Assistant Director of Research
>Protein and Nucleic Acid Chemistry Facility
>Department of Biochemistry
>University of Cambridge
>80 Tennis Court Road
>Old Addenbrookes Site
>Cambridge, CB2 1GA, UK
>Tel: +44 (1223) 333639
>FAX: +44 (1223) 766002
>e-mail: lcp2@mole.bio.cam.ac.uk
>Visit my WWW page at http://www.bio.cam.ac.uk/proj/adr/PNAC/pnac.html
-- Amos Heckendorf, Ph.D. (amos@nestgrp.com)The Nest Group, Inc., Value Added Resellers of HPLC Columns (VydacÅ, PolyLCÅ, Jordi-GelÅ, Macherey-Nagel NucleogenÅ, Higgins Analytical HAISilÅ LS, TARGAÅ & CLIPEUSÅ, and Nest Group MACCELÅ) SAI flash chromatography cartridges; and AmiKa Harvard Bioscience BioDialyserÅ, Dispo-BioDialyserÅ , 96-Well Dialysis Plates, and SuproTipÅ MS MicroSample SPE Tips.
Tel: 800-347-6378 or 508-481-6223; FAX 508-485-5736; 45 Valley Rd, Southboro, MA 01772-1306 Applications and Protocols at our NEW site: http://www.nestgrp.com/protocols/protocol.shtml
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