>> Sorry I can't be of more help. I'll be surprised if anyone gets BFP t=
o work
>> very well. I've been fooling with it for quite a while, with not very=
good
>> results.
>>We just bought the mammalian BFP expression vector pQBI50-BFP from Quant=
um
>Biotechnologies (Laval, Quebec) which features F64L, Y66H, V163A, a CMV
>promoter, neoR, and an Nhe I site just downstream from the ATG useful fo=
r
>creating fusion proteins. We'll transfect it this week and let you know=
>how things go. I was attracted to the nice Nhe I site useful for fusing=
>gfp to the C-termius of cellular proteins. We bloody hope we can get it=
>working, 'cause our real aim is FRET.
Another FRETter
I'm going in the same direction. Here is what I've learned so far
BFP from Quantum: Using the QBI50 plasmid directly for transfection of =
HeLa and CHO cells yielded poor transfection rates, but in the =
transfected cells the GFP was far brighter and more photostable than =
other UV-excitable GFPs or BFPs that I've seen (but still far from EGFP).=
=
We have recloned this BFP into our own vectors for FRET experiments and =
are currently testing them. I'm hoping for first results in the next 2-3 =
weeks. The separation from GFP(S65T) is very good (crudely measured, the =
inter-channel bleed is around 1% using our filters). I'm optimistic that =
we will be able to do decent double labelling, even if the FRET fails.
EBFP from Clontech: Using the EBFP plasmid directly for transfection of =
HeLa and CHO cells yielded either extremely poor transfection or the EBFP=
=
itself is not very bright. For whatever reason, but I've only got a few =
cells fluoresceing just above background. Attention: this was a single =
experiment. It may be all my fault. Just to make it clear: I don't know =
to what degree the different levels of fluorescence are due to possible =
differences in the amount of protein expressed.
BTW, rumour has it that there will be a first 'microscopic GFP/FRET' =
paper in one of the next issues of Nature. Look out.
Beat
-----------------------------------------------------
Dr. Beat Ludin, FMI, Maulbeerstr 66, 4058 Basel, Switzerland
Tel. +41 61 697 6697 / FAX +41 61 697 3976
Internet:ludin at fmi.ch / Compuserve:100102,1527
