m******5
发帖数: 1383 | 1 下面第二句，为什么这是个advantage? 看起来非常糟糕啊，说明在488和550左右都能
被激发
或者blue laser和green laser有别的意思？
DsRed-Express2 has additional advantages. It is excited efficiently
by both blue and green lasers that are routinely used in
flow cytometers. In bacteria, DsRed-Express2 is expressed from the
endogenous start codon much more strongly than DsRed-Express
(Supplementary Fig. 4). Another important property is phototoxicity,
which has only rarely been measured for engineered
fluorescent proteins. Our results indicate that DsRed-Express2
has low phototoxicity (Supplementary Results and Supplementary
Fig. 8 online). Thus, for whole-cell labeling, DsRed-Express2 is
the RFP of choice.
DsRed-Express2 is | i*****i
发帖数: 154 | 2 From clontech:
DsRed-Express is a rapidly maturing variant of Discosoma sp. red fluorescent
protein (DsRed). DsRed-Express fluorescent protein has enhanced solubility,
reduced green emission, and faster maturation than the original DsRed.
Although it is likely to have a tetrameric, DsRed-Express fluorescent
protein displays a reduced tendency to aggregate (1).
DsRed-Express2 fluorescent protein has even higher solubility, and was
designed for whole-cell and stem cell applications (2).
DsRed-Express excitation and emission maxima are 554 and 586 nm,
respectively
DsRed-Express2 excitation and emission maxima are 554 and 591 nm,
respectively | m******5
发帖数: 1383 | 3 The word I cited was from the paper who originally characterized DsRed
expression2, The clontech Manuel seems to have provided very different
information. I rather believe the former.
fluorescent
solubility,
【在 i*****i 的大作中提到】
: From clontech:
: DsRed-Express is a rapidly maturing variant of Discosoma sp. red fluorescent
: protein (DsRed). DsRed-Express fluorescent protein has enhanced solubility,
: reduced green emission, and faster maturation than the original DsRed.
: Although it is likely to have a tetrameric, DsRed-Express fluorescent
: protein displays a reduced tendency to aggregate (1).
: DsRed-Express2 fluorescent protein has even higher solubility, and was
: designed for whole-cell and stem cell applications (2).
: DsRed-Express excitation and emission maxima are 554 and 586 nm,
: respectively
| s******y
发帖数: 28562 | 4 我看他们也就是那么随便一说而已，没有必要把这个advantage这个词眼看得
太重。
【在 m******5 的大作中提到】
: 下面第二句，为什么这是个advantage? 看起来非常糟糕啊，说明在488和550左右都能
: 被激发
: 或者blue laser和green laser有别的意思？
: DsRed-Express2 has additional advantages. It is excited efficiently
: by both blue and green lasers that are routinely used in
: flow cytometers. In bacteria, DsRed-Express2 is expressed from the
: endogenous start codon much more strongly than DsRed-Express
: (Supplementary Fig. 4). Another important property is phototoxicity,
: which has only rarely been measured for engineered
: fluorescent proteins. Our results indicate that DsRed-Express2
| i*****i
发帖数: 154 | 5 没有仔细研究过荧光蛋白。
但是绝大部分的荧光蛋白和荧光染料如Alexa 488，594之类的都会有Excitation 和
Emission spectra，这个光谱是连续的概念。如附件一样，都是一个钟形的peak。当然
用峰值波长激发是最有效的，但是附近波长的光也会有一定程度的激发。也有一些荧光
蛋白或者染料的Excitation光谱不是非常的光滑，会形成一个类似的次要峰。 但是目
前我看到的荧光染料而言，还没有看到有两个等高的Excitation波长的。
一般来讲Excitation 和Emission的波长相距不远，488已经是标准的绿色范围了，如果
这样讲就是dsgreen，而不是dsred了。
我觉得看波普是最靠谱的，Invitrogen的molecular probe是很值得一看的。
个人愚见。
【在 m******5 的大作中提到】
: 下面第二句，为什么这是个advantage? 看起来非常糟糕啊，说明在488和550左右都能
: 被激发
: 或者blue laser和green laser有别的意思？
: DsRed-Express2 has additional advantages. It is excited efficiently
: by both blue and green lasers that are routinely used in
: flow cytometers. In bacteria, DsRed-Express2 is expressed from the
: endogenous start codon much more strongly than DsRed-Express
: (Supplementary Fig. 4). Another important property is phototoxicity,
: which has only rarely been measured for engineered
: fluorescent proteins. Our results indicate that DsRed-Express2
| b******n
发帖数: 4225 | 6 对于荧光蛋白来说，如果要做fusion，最值得考虑的是三点
1）颜色
2）发光形式是多聚还是单体。最好是单体，比如EGFP，AcGFP, mCherry，mStrawberry。
DsRed，ZsGreen等等都是臭名昭著的tetramer，做了fusion之后经常一个皿中没几个发
光的，这种发光的细胞往往对于研究是无效的（这种定位不可靠）
3）relative brightness。如果以EGFP为100，那么GFP(48)，AcGFP(82),mCherry(47)
mStrawberry(78), DsRed(176),DsRed-Express T1(58)，DsRed-Monomer(10),Zsgreen(
117)
相对而言，别的参数大部分都是浮云
如果不做fusion，尽可以选择那些相对发光强度大的比如DsRed,Zsgreen
【在 m******5 的大作中提到】
: 下面第二句，为什么这是个advantage? 看起来非常糟糕啊，说明在488和550左右都能
: 被激发
: 或者blue laser和green laser有别的意思？
: DsRed-Express2 has additional advantages. It is excited efficiently
: by both blue and green lasers that are routinely used in
: flow cytometers. In bacteria, DsRed-Express2 is expressed from the
: endogenous start codon much more strongly than DsRed-Express
: (Supplementary Fig. 4). Another important property is phototoxicity,
: which has only rarely been measured for engineered
: fluorescent proteins. Our results indicate that DsRed-Express2
| f*******e
发帖数: 628 | 7 能被不同的激光波长激发当然是个优势啊。比如你同时还要看另外一个 green 的标记
，这样只要用同一个绿的激光激发，然后两个不同 emission 颜色可以根据波长来分别
检测，可以 multiplexing。
通常 488 是指 blue, 532 是 green,在这两个波段，看光谱，的确都有不弱的激发。
但是 emission 不管是哪个 laser 激发，都还是那同样的在 600 的峰。 | m******5
发帖数: 1383 | 8 我用这个DsRed Express 2做repoter标记lineage，结果杯具了，因为我的绿光filter
是long pass……
【在 f*******e 的大作中提到】
: 能被不同的激光波长激发当然是个优势啊。比如你同时还要看另外一个 green 的标记
: ，这样只要用同一个绿的激光激发，然后两个不同 emission 颜色可以根据波长来分别
: 检测，可以 multiplexing。
: 通常 488 是指 blue, 532 是 green,在这两个波段，看光谱，的确都有不弱的激发。
: 但是 emission 不管是哪个 laser 激发，都还是那同样的在 600 的峰。
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