Our very first webinar is now live. The topic is “Fluorescent protein and sensors: A practical discussion” and you can register to view it at www.nature.com/webcasts/fluorescent_proteins. Update: Registration link inactivated. Please go here to listen to the archived discussion in .mp3 format.
Nature Methods was joined by Robert Campbell, David Piston and Thomas Knopfel who have been developing and using fluorescent proteins and sensors for years. We had a nice discussion that provided good practical information for users of these tools. If you haven’t watched it, I encourage you to do so. If you watch the webcast within the first month it is live you have the opportunity to submit questions for our participants. Please use the form on the webcast viewing page to submit questions. There will be a delay in providing answers here on our blog while we consult with the participants.
Here we will be posting the questions we receive and answers from our participants. Readers may also comment directly on the blog below but we can not guarantee that any questions asked there will be answered. We do encourage anyone in the community to chime in with their response to any questions that are posed, even if they don’t agree with our participants.
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Question from Yashaswini Balaraju
Should the fluorescent protein be cloned into the existing one?
RC: Generally speaking, you should expect only slight to modest improvements when “upgrading” from one FP (assuming it works well-enough already) to a newer variant. If a small improvement in brightness, photostability, or some other property could make a big difference to your experiment, then trying the latest variant might be worthwhile. Otherwise, it’s probably not worth the effort.
DP: It is always easiest to start with a vector that you know works, but that is not a 100% guarantee that a different FP will work exactly the same especially if switching from a jellyfish to coral derived FP.
TK: If that refers to swapping in new variants, I think we have partially covered that in the webinar. In many cases that would not be necessary or recommended unless you are ready to make many constructs (e.g. with different linkers) and screen for optimal performance.
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Question from Pradeep A R
Can you please tell me the right choice of two fluorescent proteins depending on its excitation and emission qualities and have to be used for protein interaction studies?
RC: For imaging of reversible protein-protein interactions, FRET between either a cyan and yellow fluorescent protein, or between the mRuby2 and Clover (a green/red FRET pair), are the best choices.
DP: The cyan/yellow pairs have been used the most, but optimized yellow/red pairs (mClover/mRuby2) are probably the better choice going forward.
TK: Depends on mechanism of interest? FRET, complementation, dimerisation?
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Question from Paulo Saldanha
I would like to know how can I choose a fluorescence protein that should bind my human smooth muscle cell membrane and can be light resistant for experiences for about 2 hours in microscopy and should resist to fluorescence photos every minute or so for about the 2 hours.
RC: EGFP is one of the most photostable fluorescent proteins and would be a good choice for an experiment such as this.
TK: EGFP/Clover with trafficking signal and lipid anchor.
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Question from Mohamed Abou Attia
I am plant pathologist but I do not know how to use fluorescent proteins in plant pathology.
RC: A variety of colors of fluorescent proteins have been used successfully in plants, and a number of reviews have been written on the topic. Once challenge is that chlorophyll fluorescence can obscure the fluorescence from red fluorescent proteins, so cyan, green and yellow FPs may generally be preferred.
TK: FP have been successfully used in plants. Codon optimization is helpful. This works in healthy plants, so I guess it also will work when plants are in a pathological condition.
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Question from Jamila Oppong
I would like to know how to calculate the FRET and also how to effectively process the images of a photobleaching experiment.
RC: Chapter 13 of Principles of Fluorescence Spectroscopy by Joseph R. Lakowicz has a very thorough explanation of FRET principles and calculations of FRET efficiency. In a nutshell, you need to record the fluorescence intensity in the donor channel before (FDA) and after (FA) photobleaching of the acceptor. You can then calculate the FRET efficiency E = 1 – FDA/FA.
DP: We also published this along with the jellyfish FP R0 values in Patterson et al. 2000. Anal. Biochem. 284:438-440.
TK: https://en.wikipedia.org/wiki/F%C3%B6rster_resonance_energy_transfer
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Question from Dillip Kumar Bisoyi
Do you all have any idea about the fluorescence nature of alpha-Lactalbumin and beta-Lactalbumin? Please elaborate me.
RC: no
DP: me neither
TK: pass
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Questions from Amin Karmali
How do you immobilize these proteins for biosensor development? Which support can you use and which experimental conditions
RC: Immobilization of FPs on solid surfaces is not something that is commonly done, since the main advantage of FPs relative to other fluorophore technologies (i.e., synthetic dyes and quantum dots) is that they are genetically encodable. That said, my group has had success at biotinylation of FPs and immobolization on streptavidin coated surfaces. Please see McEvoy et al. PLoS ONE 7(12): e51314.
DP: We have used microdroplets and immobilizationn in gels for this. The microdroplet procedure is described in Kremers &. Piston. 2010. J. Vis. Exp. Jun 26(40) pii:1995 and the gel immobilization in Piston et al. 1999. Methods in Cell Biology, volume 58 Chapter 3, pp. 31-48.
TK: Why do you want to immobilize?
I am working on FLIPglucose600micro and the glucose binding to this protein does not show an increase in fluorescence at 482nm. Why, please?
RC: Many of the sensors based on periplasmic binding proteins exhibit very small FRET changes. A glance at Figure 1 of the paper that first describes this indicator (J. Biol. Chem. 2003, 278:19127-33) confirms that this is certainly true in this case. If the concentration is low, the real FRET change could be lost in the noise.
TK: ask author: mailto:wfrommer@stanford.edu
Which are the best immobilization supports for FLIPglucose sensors and which chemistry must be used in order to reduce their inactivation as far as fluorescent properties are concerned?
RC: You could use biotinylation as suggested above, though probably the easiest method is to immobilize to a Ni-NTA modified surface through a 6-his tag appended to the sensor. Since they are being used in vitro, you might be able to prevent photobleaching by degassing your buffers or adding antioxidants to the solution.
TK: as above