发信人: Ras (很颓的猪。), 信区: Biology
标 题: Re: 请教:GFP and its variants from clontech
发信站: The unknown SPACE (Thu Jun 8 10:46:12 2000), 转信


【 在 SoundingSea (昨日重现) 的大作中提到: 】
: Hi, is there anyone who is familiar with GFP/EGFP(Green
: Fluorescent Protein,
: living color series from Clontech)? I wanna clearify the
: followings before
: I try it.
: 1)How long does it take for GFP to be expressed/become
: fluorescent?
: (I am looking for sth that can be expressed instantly/within
: few mins
: once the constructs are introduced into cells)

Normally it takes 3-5 hours after transfection to express enough GFP protein
in cells to get detectable fluorescence. Another trick is that it will take
about 3 hours for the fluorophore on GFP to be oxidized so that it can be
fluorescent. As far as I know, the quickest detection of GFP (or other
living fluorescent variants from Clontech) is around 8 hours after
introduction of DNA. The earliest time I ever tried is 10 hours after
transfection, but it is weak.

However, if you really want to see the fluorescence quick, you may try
introduce mRNA, instead of DNA, into the cells (by electroporation etc.).
It should be much quicker, but still not in the range of minutes.

: 2)does it need incubation?
: (I do in situ single cell lever gene introduction, can't
: incubate on site
: at this stage)

Don't quite understand ur question. But I guess it doesn't need incubation
since it is the so called "living color protein" which means you can see
the fluorescence even in live cells (withou any treatment).

: Suggestions on other kind of vectors are also highly
: appreciated.

Heard about some other vectors, such as Super Cylone GFP. But none of them
are as good as pEGFP from Clontech. I have 2 suggestions for you:
1. try making a inducible construct. It may produce protein quicker upon
induction.
2. Try place multiple GFP coding sequence into your construct. By this way
the fluorescense should be much stronger and be visible faster.

(btw, if u have any further questions, u can mail me here)

: Thanks much!
: s.s.


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