Measuring Fluorescene of RFP - (Jun/17/2013 )
I was wondering if someone with more knowledge than me in the area of measuring fluorescent intensity of fluorescent proteins can shed some light on this new topic. I am a newbie in this field so I don't know precise terminology but I will try to make sense:
I have transformed bacteria containing a plasmid with a promoter-RFP (red fluorescent protein) fusion that I am using as a reporter of gene activity. The RFP is dsRed from dsRed-Express2 plasmid, and it has an excitation maximum of 554nm and an emission maximum of 591nm. My question is do I need to set the excitation and emission values exactly on the machine (a PerkinElmer Victor x5 multilabel plate reader)? Is there one that I must set exactly right?
We currently don't have filters that exactly match those numbers so I am running my experiments without them but I am not getting good results. I wanted to know if the settings were the problem or some other aspect of my experiment may be it.
You don't need filters that match either exactly, but if you have one or both, it will be helpful easier. In your current situation you need to look at the excitation and emission spectra and see where your filters lie and how this will affect the amount of light that reaches the sensor. For example if you look at the excitation spectrum and see that the filter you are currently using will only give you 70% of the maximum, then that is the maximum you could expect to get out. Likewise for the emission. A combination of the two will be multiplicative (e.g. 50% emission of 70% excitation) rather than subtractive (70%-50%) (that's presuming I have my maths correct).
It would also pay to check what sort of spectrum the filters will let through - are they specific for a narrow bandwidth or are the long pass or short pass?
In your current situation, you may find it best to either use only the excitation or the emission filters if they are both narrow spectrum to maximize one of the parameters so that you either get maximal input or maximal output. Personally I would go for maximal excitation (i.e. just a lens rather than a filter) and have a specific emission filter.
Thank you for your reply Bob.
I have seen in some articles that the values were matched exactly and then in other resources I found that the excitation was matched exactly while the emission value was a ballpark range.
I am beginning to believe that perhaps another part of my experiment is faulty.
Hi Ted, I would like to ask you if you have got better results. I have recently managed to make a construct using the same vector and am gathering approaches before I jump into the assessment of expression.
Ted checks in occasionally - his profile says last active march 12, so you might be waiting a while for an answer.
Hey Bob1 Maybe you can help here, I have succeeded to fuse two bacterial genes(one is a transcriptional activator and the other a protein coding gene). the genes were as well cloned into pDsred-Express 2. The genes were amplified including their start codon and without the stop codon for the first but the second contains its stop codon and they were cloned upstream of the dsred(hoping to use it as a reporter gene). Now I have these questions:
1. Do you think the genes are well placed for the use of dsred as a reporter for their expression?
2. any tip about preparing my sample prior being viewed on a fluorescent microscope?
If you were intending to make fusion proteins the one with the stop codon will probably not encode the RFP as the stop codon should prevent this from being transcribed.
These proteins are naturally fluorescent, there shouldn't be much that you need to do to get your samples to fluoresce other than using the correct wavelength for excitation. However, some fixation methods (e.g. formaldehyde) can quench fluorescence of GFP, so as most of the other colours are derivatives of GFP, they will most likely also be quenched by fixing.
Thank you, I have also prepared the fused genes without the stop codon on the3'of the second gene(to be on the safe side, I will try both). Am intending to assess some chemicals using this construct; am a bit nervous but I hope I will get expression!
Thanks Bob1, I will let you know the outcome.