请教个实验中遇到的问题, 关于characterize the location of a protein in the cell

seamless_skyUID: 2228384

My undergraduate thesis is to characterize the cellular location of a novel protein.
The sequence of the gene is known. The gene can be successfully expressed in PC12 cells. Imaging study has shown it is located close to membrane, but we do not know whether it is transmembrane. Looks like it has a tyrosine phosphorylation site. It can be glycosylated (speculation).
我想问一下有没有什么办法在不知道很多protein property的情况下测试protein's location in the cell. 没有时间和资源做很多trial and error. 有什么computer program可以通过amino acid sequence测出protein是不是transmembrane的吗? 我在molecular方面没有什么经验. 但是Supervisor似乎要偶自力更生.
:L
多谢

hermeanvvUID: 2180253

偶是外行...
是否可以用另一个荧光染料标记一个跨膜蛋白,而后两张图merge一下?

seamless_skyUID: 2228384

多谢。 我也是外行。以前没有做过分生的东西。
按照你说的话需要co-expression two vectors. 不知道结果是不是能说明问题啊。It may depend on where the fluorescent protein is attached
需要问一下他们以前的imaging是怎么做的。

seamless_skyUID: 2228384

原帖由 neurogenomics 于 2006-10-19 06:30 发表
with the gene sequence being known, you can get the hypothetical amino acid sequence, then you may analzye the hydrophobicity of the hyphothesized protein, a hydrophobic region of enough length is  ...

Thanks. Is there suitable computer program for predicting a transmembrane segment? I know the amino sequence already and am thinking in the same way.

Not familiar with the use of SEm for this purpose. Need to read more on that.

[ 本帖最后由 seamless_sky 于 2006-10-19 12:26 编辑 ]

seamless_skyUID: 2228384

再问一个naive question: Do all transmembrane segments consist of alpha-helix?

seamless_skyUID: 2228384

Thanks so much! Dearly appreciate your advice and suggestions.
I found some computer programs to do the preliminary analysis on Expasy (transmembrane domain, signal peptide etc). The result is dubious. When I reported it to my supervisor, he said he obtained the same thing (obviously wanted me to Do It Myself purposely), yet he thinks it is worth doing further testing. He thinks it can be a special type of receptor that has single pass and very large intracellular and extracellular components. He also thinks there might be a special rER signal peptide that is not in the data base. Sigh... I am too naive.
There is no SEM facility available. I guess confocal results are not convincing enough. So have to continue with the trial and error biochemical testing ie. to label surface proteins. Hopefully PC12 cells will not be contaminated again. Fingers crossed.