发信人: royluo ( 罗马情结), 信区: Biology
标 题: Re: deglycosylation problem! please help
发信站: BBS 未名空间站 (Tue May 15 00:15:06 2007)

For PNGase F digestion, the protein has to be fully denatured in 1%SDS+1%
beta-ME before digestion ; for Endo H digestion,
denaturation is not necessary, but it wouldn't hurt. Did you include
a positive control? If not, you can try ovalbumin, which
contains high-mannose N-glycan so that both enzyme
will cut.

I am also attaching my protocol here.

(1)Precipitate protein sample through acetone precipitation.

(2)redissolve the protein pellet in 25ul 1%SDS + 1% beta-ME,
boil for 5 minutes.

(3)Add 225ul PNGase F digestion buffer(50mM Tris-Hcl, pH8.6,
10mM EDTA, 0.7%(w/v) NP40 and Protease Inhibitor Cocktail)
or EndoH digestion buffer (100mM sodium Acetate pH5.0 with
Protease Inhibitor Cocktail). This step is essential! The sample is
diluted 10 fold because PNGase F and Endo H can only work in
non-denaturing conditions.

(4)Add PNGase F or Endo H. For PNGase F, incubate at RT for
more than 15 hrs; for EndoH, incubate at 37 degree for more
than 15 hrs.

(5)Precipitate the protein sample again through acetone
precipitation.

(6)Analyze the sample by SDS-PAGE.
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