In article <32233928.6B4A at bioneer.kaist.ac.kr>, Àå°ÇÈñ
<kernee at bioneer.kaist.ac.kr> wrote:
> Dear Netters..
>> What would be the best way to cleave and recover only protein
>> from complex glycoprotein ?
...... (SNIP) ......
As the other poster points out, PNGase F will not remove N-linked glycans
with Fuc-alpha-1,3- linkages in the core. However, PNGase A, from
_A_lmonds will hydrolyze these ologosaccharides. It is commercially
available from Seikagaku Corp.
One consideration that you should be aware of is that your glycoprotein
may have O-linked sugars in addition to, or instead of, the N-linkeds,
which cannot be completely removed without destroying part or all of your
peptide backbone. Perhaps you could do some additional characterization by
performing lectin blots of your glycoprotien on PVDF membranes. This can
be done with ng of material and, if the right lectins are chosen, can
provide a lot of information. A monosaccharide analysis would also be
useful.
For more information, check out a 'how to' book such as
"Guides to Techniques in Glycobiology" (1994) Lennarz and Hart
(eds.), Mtds in Enzymol. Vol 230, various chapters.
Good Luck! Rob Trimble, NYSDOH, Albany NY.
