Measuring Fluorescene of RFP - (Jun/16/2013 )
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.