Date: 6 Aug 1997 06:48:08 -0700 From: Beat Ludin To: fluorpro@net.bio.net Subject: Re: BFP anyone? >> Sorry I can't be of more help. I'll be surprised if anyone gets BFP to >> 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 > Quantum Biotechnologies (Laval, Quebec) which features F64L, Y66H, > V163A, a CMV promoter, neoR, and an Nhe I site just downstream from the > ATG useful for 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. Beat Ludin writes: 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@fmi.ch / Compuserve:100102,1527