发信人: leohawk (leohawk), 信区: Biology
标 题: Re: 关于characterize a protein的问题求助
发信站: The unknown SPACE (Sun Nov 10 16:13:21 2002), 转信

my understanding of your question is:

the protein you are interested in, say LHRH, has N terminal sequence
identical as protein A. Now you want to use RT-PCR to get the cDNA for
this protein.

I am not sure if protein A is also in your organism, if not, RT-PCR might
work, if yes, RT-PCR won't work as you will get cDNA for protein A...

I personally think you should try two methods, maybe 3 to be sure:

1. RT-PCR.
2. cDNA library
3. Genomic library.

method one is easier to try, but hard to be sure, method 2 is more reliable,
risk is cDNA for your protein is of low abundance, method 3 will definitely
work.

in library screening, protein A's sequence can be used as probe.

do this only after your database search fail to give you any information.

screening library is only 1 weeks work... if you have library handy.


【 在 lhrh (never ever) 的大作中提到: 】
: 【 在 leohawk (leohawk) 的大作中提到: 】
: : not sure if the organism's genome is done, otherwise, you will have a clear
: : cut answer. my guess is there is not much info on it right now for you.
: : I don't think what you are about to do is going to conform this protein is
: : protein A, it can only tell you you can get protein A using this method.
: ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
: What's the meaning of your last sentence? With 2D gel, I can get protein A,
: but I can't confirm it's protein A? Or, with RT-PCR, I can get nucleic acid
: sequence for protein A, but I can't know the protein I obtained is protein A?
: I am really confused. Can you tell sth. in detail. Is there any obvious flaw
: in the idea of RT-PCR?
: Thanks


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