Susanne,
Yes, the Pierce method is a colorometric assay at 550 nm. The
first step is periodate oxidation followed by addition of 1 volume of
their glycoprotein detection agent (0.5% solution in 1N NaOH) which
gives a purple color after 1 hour incubation. The assay does give the
purple color with their glycoprotein standards (Ovalbumin,
Apo-Transferrin, Fetuin, alpha-1-Acid Glycoprotein)and galactose but
even after 1.5 hours the absorbance at 550 nm continues to rise. This
makes it impossible to get a standard curve. We would like to be able to
assay for galactose and lactose conjugates and perhaps in the future
more complicated polysaccharides. Got any suggestions or insights?
Thanks for the help!
Bruce.
Bruce Frank, Ph.D.
Director, Chemistry
Kimeragen, Inc.
300 Pheasant Run
Newtown, PA 18940
Phone: 215-504-4444 x214
Fax: 215-504-4545
email: bfrank@kimeragen.com
-----Original Message-----
From: Susanne.Kavitski@aventis.com [mailto:Susanne.Kavitski@aventis.com]
Sent: Tuesday, March 28, 2000 10:04 AM
To: Bfrank@Kimeragen.com
Subject: RE: Sugar assay
Frank,
Although I have no experience with the Glycoprotein Carbohydrate
Estimation Kit from Pierce, I have many years working with sugar
conjugates
and corresponding sugar assays. A good, old reference book is
Experimental
Immunochemistry, by Kabat and Mayer's. What exact sugar(s) are you
attempting to quantitate? Based on what your monosaccharide is, I might
be
able to suggest the appropriate assay to use. I've used sodium
meta-periodate to cleave vicinal hydroxyl groups in sugars; the
resulting
product is a reducing sugar, i.e. aldehyde. Is Pierce's method a
colorimetric assay; if so, at what wavelength is the chromophore
measured?
Another more complicated method used for sugar quantitation is
via
ion exchange chromatography using an electrochemical detector (i.e.
Dionex).
Susanne
> -----Original Message-----
> From: Frank Bruce [SMTP:Bfrank@Kimeragen.com]
> Sent: Monday, March 27, 2000 11:33 AM
> To: Recipients of ABRF List
> Subject: Sugar assay
>
> We have been trying to set up sugar assays for sugar-conjugates of
> polymers, lipids, proteins, etc. One conjugate we prepared using
> galactosyl phenylisothiocyanate does not give a positive test using
> resorcinol/sulfuric acid. We just purchased a Glycoprotein
Carbohydrate
> Estimation Kit from Pierce which uses sodium meta-periodate and a
> proprietary "glycoprotein detection reagent" to measure sugar content
in
> these and other samples. Does anyone have experience with this kit and
> any comments on its accuracy and reproducibility? Do you have a guess
on
> what the detection reagent is? Are there other sugar assays that might
> be of some use? Thanks in advance for the help!
>
> Bruce.
>
> Bruce Frank, Ph.D.
> Director, Chemistry
> Kimeragen, Inc.
> 300 Pheasant Run
> Newtown, PA 18940
> Phone: 215-504-4444 x214
> Fax: 215-504-4545
> email: bfrank@kimeragen.com
>
>
> -----Original Message-----
> From: Beth Fowler [mailto:beth_fowler@leukosite.com]
> Sent: Friday, March 24, 2000 8:48 AM
> To: Recipients of ABRF List
> Subject: Fwd: Computer Virus You Might Want to Know About
>
>
>
> >Date: Fri, 24 Mar 2000 08:41:52 -0500
> >From: John Kelly <kelly@mpi.com>
> >X-Mailer: Mozilla 4.51 [en] (WinNT; U)
> >X-Accept-Language: en
> >To: Group All Millennium <millennium-all@mpi.com>
> >Subject: Computer Virus You Might Want to Know About
> >
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> Elizabeth Fowler, Ph.D.
> Director, Analytical Development and Quality Control
> Millenium Pharmaceuticals, Inc.
> 75 Sidney Street
> Cambridge, MA 02139
> Phone: 617-551-3730
> FAX: 617-551-3745
> beth_fowler@leukosite.com
> efowler@ma.ultranet.com
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