y******n
发帖数: 8667 | 1 用TIRF or fluorescence microscopy来研究tubulin dynamic in vitro regulated by
other tip proteins.
But now I met a lot of problems:
1. Is labeled tubulin and tubulin easy to lost their activity in vitro?
2. What conditions is better for their polymerization?
以前看到过很好的GMPCPP seeds， 但之后再也重复不出来了。同一批labeled的
tubulin蛋白好象都形成一些很亮的点，应该是蛋白凝聚了或变性了。但新labeled 的
tubulin 也不行。有人有什么建议吗？ |
l**********1
发帖数: 5204 | 2 GTP→GDP already
please go to new book
"New Insights into the Mechanisms
of Cytomotive Actin and Tubulin
Filaments" 2011 Elsevier Inc.
by Christopher H.S. Aylett, Jan Lo¨we, and Linda A. Amos
full text link:
//www2.mrc-lmb.cam.ac.uk/groups/JYL/PDF/AmosReview2011.pdf
its pp20
On phosphate release, Wang and Nogales proposed that the contact
between T7 and the nucleotide of the adjacent subunit within the E-site
interface becomes disrupted, leaving contacts only on the side of the
subunit–subunit interface further from the nucleotide, therefore placing it
in a fully curved-like state. The effect of nucleotide hydrolysis would
therefore be to disrupt the subunit–subunit interface and favor a less stable
form of the tubulin protofilament.
ignored.
by
的
【在 y******n 的大作中提到】
: 用TIRF or fluorescence microscopy来研究tubulin dynamic in vitro regulated by
: other tip proteins.
: But now I met a lot of problems:
: 1. Is labeled tubulin and tubulin easy to lost their activity in vitro?
: 2. What conditions is better for their polymerization?
: 以前看到过很好的GMPCPP seeds， 但之后再也重复不出来了。同一批labeled的
: tubulin蛋白好象都形成一些很亮的点，应该是蛋白凝聚了或变性了。但新labeled 的
: tubulin 也不行。有人有什么建议吗？
|
l**********1
发帖数: 5204 | 3 Continue:
please look a lo below figure
cited from upper floor that E-Book.
【在 l**********1 的大作中提到】
: GTP→GDP already
: please go to new book
: "New Insights into the Mechanisms
: of Cytomotive Actin and Tubulin
: Filaments" 2011 Elsevier Inc.
: by Christopher H.S. Aylett, Jan Lo¨we, and Linda A. Amos
: full text link:
: //www2.mrc-lmb.cam.ac.uk/groups/JYL/PDF/AmosReview2011.pdf
: its pp20
: On phosphate release, Wang and Nogales proposed that the contact
|
s****n
发帖数: 234 | 4 Hope these will help:
http://mitchison.med.harvard.edu/protocols/poly.html
http://hymanlab.mpi-cbg.de/hyman_lab/methods/tubulin/tubulin_pr
http://www.mpi-cbg.de/nc/research/research-groups/joe-howard/pu (look for "Microtubule dynamics reconstituted in vitro and imaged by single-molecule fluorescence microscopy")
BTW: what TIPS proteins?
by
的
【在 y******n 的大作中提到】
: 用TIRF or fluorescence microscopy来研究tubulin dynamic in vitro regulated by
: other tip proteins.
: But now I met a lot of problems:
: 1. Is labeled tubulin and tubulin easy to lost their activity in vitro?
: 2. What conditions is better for their polymerization?
: 以前看到过很好的GMPCPP seeds， 但之后再也重复不出来了。同一批labeled的
: tubulin蛋白好象都形成一些很亮的点，应该是蛋白凝聚了或变性了。但新labeled 的
: tubulin 也不行。有人有什么建议吗？
|
l**********1
发帖数: 5204 | 5 Are you sure you can use only fluorescence microscopy来研究tubulin dynamics
in vitro regulated by other tip protein?
http://www.mitbbs.com/article_t1/Biology/31629913_0_2.html
27th floor
If not,
please just go to
//cryoem.berkeley.edu/microtubules.html
//cryoem.berkeley.edu/cryoem
//cryoem.berkeley.edu/microscopes
more just click all icons you can open that web sites.
//mcb.berkeley.edu/index.php?option=com_mcbfaculty&name=nogalese
then rethink about which kind of instrument will be needed?
by
的
【在 y******n 的大作中提到】
: 用TIRF or fluorescence microscopy来研究tubulin dynamic in vitro regulated by
: other tip proteins.
: But now I met a lot of problems:
: 1. Is labeled tubulin and tubulin easy to lost their activity in vitro?
: 2. What conditions is better for their polymerization?
: 以前看到过很好的GMPCPP seeds， 但之后再也重复不出来了。同一批labeled的
: tubulin蛋白好象都形成一些很亮的点，应该是蛋白凝聚了或变性了。但新labeled 的
: tubulin 也不行。有人有什么建议吗？
|
s****n
发帖数: 234 | 6 I think the in vitro tubulin dynamics experiments using TIRF microscopy will
be able to say whether the presence of tip protein(s) affects the tubulin
rescue and shrink rates. A complete understanding of the underlying
molecular mechanism may require structural studies using other methods
including EM.
dynamics
【在 l**********1 的大作中提到】
: Are you sure you can use only fluorescence microscopy来研究tubulin dynamics
: in vitro regulated by other tip protein?
: http://www.mitbbs.com/article_t1/Biology/31629913_0_2.html
: 27th floor
: If not,
: please just go to
: //cryoem.berkeley.edu/microtubules.html
: //cryoem.berkeley.edu/cryoem
: //cryoem.berkeley.edu/microscopes
: more just click all icons you can open that web sites.
|
l**********1
发帖数: 5204 | 7 Exactly your opinion is right.
plus even without CryoEM below group still can do simulation with modeling for their most possbile
mechanism discovery
please refer the figure G its scale bar 200 nm it is below florescent microscopy resolution limits,
so they cannot resolve the fusion of the individual FIP3-endosomes.
cited from
//www.ncbi.nlm.nih.gov/pubmed/21486954
and
//www.ncbi.nlm.nih.gov/pubmed/16818889
will
【在 s****n 的大作中提到】
: I think the in vitro tubulin dynamics experiments using TIRF microscopy will
: be able to say whether the presence of tip protein(s) affects the tubulin
: rescue and shrink rates. A complete understanding of the underlying
: molecular mechanism may require structural studies using other methods
: including EM.
:
: dynamics
|
