GFP

Bernard Murray bernard at elsie.nci.nih.gov
Mon Apr 28 01:09:12 EST 1997


In article <3360aac1.81756201 at news.ki.se>, csakis at ki.se says...
>Hi!
>I constructed a tetracycline regulatable vector system. I am checking
>the regulation by GFP. I do not have very good experience with
>microscopy and cell work. Can anybody suggest me some good protocols
>how to look at cells? Is it better with live cells or fixed cells? And
>such things.
>Thanx
>Csaba
>csakis at ki.se

Have a look at bionet.molbio.proteins.fluorescent as there's a long
running/recurring thread on this topic.  From personal experience
if you are using a human codon optimised GFP variant and have a decent
promoter you should be able to see it in live cells under conditions
where you'd normally look at fluorescein.  I found that it helped
to switch to medium without phenol red but that's my preference.
As far as fixation is concerned avoid ethanol and try light fixation
in formaldehyde or paraformaldehyde.
	If you have a look at the Clontech web site they have an on-line
version of their GFP vector manuals and these have a lot of tips on
sample preparation.
		Bernard

[No relationship to Clontech except as a customer]

Bernard Murray, Ph.D.
bernard at elsie.nci.nih.gov (National Cancer Institute, NIH, Bethesda MD, USA)




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