BFP anyone?

Beat Ludin b_ware at COMPUSERVE.COM
Wed Aug 6 08:48:08 EST 1997


>> 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




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