x****u
发帖数: 530 | 1 GFP表达之后，容易被降解吗？
如果一个蛋白融合GFP之后，蛋白降解是否一定会导致GFP也一起降解了？ |
s******s
发帖数: 13035 | 2 GFP非常稳定，通常不稳定的蛋白和GFP fuse以后都会变稳定。
降解以后也常常还留着GFP，你跑下wb就知道了
【在 x****u 的大作中提到】
: GFP表达之后，容易被降解吗？
: 如果一个蛋白融合GFP之后，蛋白降解是否一定会导致GFP也一起降解了？
|
b******s
发帖数: 1089 | 3 请问这个有很多证据吗？如果这样，那么GFP fuse的蛋白定位岂不是不够准确。很多是
蛋白降解后剩下的free GFP？
【在 s******s 的大作中提到】
: GFP非常稳定，通常不稳定的蛋白和GFP fuse以后都会变稳定。
: 降解以后也常常还留着GFP，你跑下wb就知道了
|
a***y
发帖数: 19743 | 4 GFP单独主要分布在细胞质和细胞核。
【在 b******s 的大作中提到】
: 请问这个有很多证据吗？如果这样，那么GFP fuse的蛋白定位岂不是不够准确。很多是
: 蛋白降解后剩下的free GFP？
|
s******s
发帖数: 13035 | 5 我说很多，是指有很多这样的情况，不是指GFP的百分比很多。
当然,GFP fuse的蛋白定位有很多问题。通常的解决办法是做KO，
然后用GFP fusion rescue表型。不过严格来说，就算localization
不太对，如果GFP fusion表达量很大的话，有些时候也能rescue
【在 b******s 的大作中提到】
: 请问这个有很多证据吗？如果这样，那么GFP fuse的蛋白定位岂不是不够准确。很多是
: 蛋白降解后剩下的free GFP？
|
s******y
发帖数: 28562 | 6 这种情况其实不普遍。
另外，如果融合上的蛋白是被各种蛋白酶来降解的话，的确常常会留下一个孤零零的
GFP, 但是如果是被proteasome 降解的话一般会把GFP也同时降解了。
【在 b******s 的大作中提到】
: 请问这个有很多证据吗？如果这样，那么GFP fuse的蛋白定位岂不是不够准确。很多是
: 蛋白降解后剩下的free GFP？
|
c**********o
发帖数: 69 | 7 GFP稳定的证据就是CD上了90度还是beta-sheet,看不出random coil的潜质。。。
【在 b******s 的大作中提到】
: 请问这个有很多证据吗？如果这样，那么GFP fuse的蛋白定位岂不是不够准确。很多是
: 蛋白降解后剩下的free GFP？
|
Q*****n
发帖数: 4546 | 8 强悍
【在 c**********o 的大作中提到】
: GFP稳定的证据就是CD上了90度还是beta-sheet,看不出random coil的潜质。。。
|
x****u
发帖数: 530 | 9 谢谢大家的回复。
下面这篇文章是说GFP half life是2.8h吗？
Automated live cell imaging of green fluorescent protein degradation in
individual fibroblasts.
Halter M, Tona A, Bhadriraju K, Plant AL, Elliott JT.
Source
Abstract
To accurately interpret the data from fluorescent proteins as reporters of
gene activation within living cells, it is important to understand the
kinetics of the degradation of the reporter proteins. We examined the
degradation kinetics over a large number (>1,000) of single, living cells
from a clonal population of NIH3T3 fibroblasts that were stably transfected
with a destabilized, enhanced green fluorescent protein (eGFP) reporter
driven by the tenascin-C promoter. Data collection and quantification of the
fluorescence protein within a statistically significant number of
individual cells over long times (14 h) by automated microscopy was
facilitated by culturing cells on micropatterned arrays that confined their
migration and allowed them to be segmented using phase contrast images. To
measure GFP degradation rates unambiguously, protein synthesis was inhibited
with cycloheximide. Results from automated live cell microscopy and image
analysis indicated a wide range of cell-to-cell variability in the GFP
fluorescence within individual cells. Degradation for this reporter was
analyzed as a first order rate process with a degradation half-life of 2.8 h
. We found that GFP degradation rates were independent of the initial
intensity of GFP fluorescence within cells. This result indicates that
higher GFP abundance in some cells is likely due to higher rates of gene
expression, because it is not due to systematically lower rates of protein
degradation. The approach described in this study will assist the
quantification and understanding of gene activity within live cells using
fluorescent protein reporters. |
i***l
发帖数: 1656 | 10 i have hands on experience on this, correct, fusion protein with gfp tag is
usually more stable that itself
of course you should confirm the stability when you do subcellular
localization
GFP fuse的蛋白定位 only when the fusion is stable, commensense ba
【在 b******s 的大作中提到】
: 请问这个有很多证据吗？如果这样，那么GFP fuse的蛋白定位岂不是不够准确。很多是
: 蛋白降解后剩下的free GFP？
|
|
|
O******e
发帖数: 4845 | 11 我比较好奇，你这个加GFP后蛋白变得更稳定的结论是怎么得出来的？测了半衰期了？
考虑了过表达的因素了么？
is
【在 i***l 的大作中提到】
: i have hands on experience on this, correct, fusion protein with gfp tag is
: usually more stable that itself
: of course you should confirm the stability when you do subcellular
: localization
: GFP fuse的蛋白定位 only when the fusion is stable, commensense ba
|
s******y
发帖数: 28562 | 12 他们用的不是普通的eGFP, 而是destablized eGFP
【在 x****u 的大作中提到】
: 谢谢大家的回复。
: 下面这篇文章是说GFP half life是2.8h吗？
: Automated live cell imaging of green fluorescent protein degradation in
: individual fibroblasts.
: Halter M, Tona A, Bhadriraju K, Plant AL, Elliott JT.
: Source
: Abstract
: To accurately interpret the data from fluorescent proteins as reporters of
: gene activation within living cells, it is important to understand the
: kinetics of the degradation of the reporter proteins. We examined the
|
i***l
发帖数: 1656 | 13 sure, i 'd share. but remember every protein is different
at least it worked for me once.
i was trying to purify a membrane protein, which did not give me full length
on protein gel after purification, and the fusion protein (N term gfp) gave
me at least partial full length fusion, based on the size in sds-page.
every other things were the same, same expression system, same induction
conditions, same extraction method.
but we still could not get enough for crystalization, then gave up, hehe
【在 O******e 的大作中提到】
: 我比较好奇，你这个加GFP后蛋白变得更稳定的结论是怎么得出来的？测了半衰期了？
: 考虑了过表达的因素了么？
:
: is
|
i***l
发帖数: 1656 | 14 good catch
【在 s******y 的大作中提到】
: 他们用的不是普通的eGFP, 而是destablized eGFP
|
i***l
发帖数: 1656 | 15 maybe i should not use "usually", coz it worked for my specific protein
【在 O******e 的大作中提到】
: 我比较好奇，你这个加GFP后蛋白变得更稳定的结论是怎么得出来的？测了半衰期了？
: 考虑了过表达的因素了么？
:
: is
|
