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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