发信人: sunnyday (飞鸟), 信区: Biology
标 题: Re: 跨膜蛋白的orientation?
发信站: The unknown SPACE (Thu Dec 26 12:33:17 2002) WWW-POST

【 在 odz (odd) 的大作中提到: 】
: How can I know there is a signal peptide in te pre-mature protein?
Search the amino acid sequence on data base for signal peptide.
MOst signal peptides have been characterized.
:
: Could you please explain the protease K protection experiment and
: the glycosylation test in detail?
I think, roughly, but not in detail: digest the whole cell or the membrane
vesicles make from supersonic, without using any detergent. If a pepetide is
outside membrane, it will be digested. If it's inside, it will not.
GFlycosylation should use similar mechanism, but instead of protease,
glycosylase is used to labled the outside peptide, then a mess spec can be
used to determined the labled site.

:
: below are some more information about my protein:
:
: In my protein there are 8 EGF repeats at around 300 aa. position. EGF
repeats
: are supposed to be outside of the cell. Two opposite opinions exist
concerning
: the orientation of the protein, typeI and typeII. The putative TM domains
are
: thought to be before and after the EGF repeats in these two type of
proteins,
: respectively.
:
: Thanks!
:
:
: 【 在 akite (找路的人) 的大作中提到: 】
: : firstly, if there is a signal peptide in the pre-mature protein,
definately
: : N-ter is outside, if there is not, positive charge between the N-terminal
Met
: : and the first TM segment generally makes the N-ter inside, negative charge
: : makes it outside.
: : other ways: 1. protease K protection experiment; 2. glycosylation test
: : 【 在 odz (odd) 的大作中提到: 】
: : : 用什么方法能determine 一个蛋白的N端在膜外还是在胞内？
: : : 我的蛋白有300 KD, 是个跨膜蛋白。
: : : 我想到的方法是在体外培养的细胞中， 表达N 或 C 端fusion protein.
: : : then use FACS to cells whose fusion protein has its epitope outside.
: : : anyone has good suggestions? Thanks!
:
:


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