Hi Dan,
Thank you.
I read the paper and I wish there were more such a study with more promoters other than vit-2P and hsp-90P (if we think there is an interaction between the intron and the promoter), also a study to show the non-functional mCherry products (dark and no-fluorescent, but junk aggregates) due to increased number of introns.
I think that one of the sources of aggregation can be those two extra introns (or even all three), that potentially cause alternative splicing and misfolded isoforms.
Hi bdackley,
Thank you very much for insight and information and for directing me to FPbase.org which is a great resource.
My main problem is aggregation now. The brightness is not as concerning.
…“mCherry often gets mislabeled as an aggregating tag, but that’s likely due to the fact that it is still fluorescent inside lysosomes”…
In my hand it is not only in lysosomes (Lysosome: I guess according to the literature- I haven’t labeled lysosome personally yet), LROs (I use additional green and DAPI filter to distinguish them), but also these RED-FPs are diffuse cytoplasmic, and some threadlike structures, all membranes sometimes, plus huge irregular aggregation (I have tried 0.5-1ng/ul to 2, 3, 5, 10, 15, 25, 50-100 ng/ul, even higher concentrations and see them especially with higher ng/ul of the injected construct) that they don’t resemble as a subcellular organelle or a round well-shaped lysosomal structure or any structure.
Neither I see the expected structure to be brighter and sometimes it is totally missing as if the RED-FP is everywhere except where it must be.
Unfortunately, I don’t have the resource to investigate these Red-FPs vigorously and I wish a resourceful lab could investigate them more to offer it to C. elegans investigators. I will clone different current ones: Scarlet-I, Fusion-Red (has only one 5’-intron) and mNeptune under a promoter and 3’UTR that has been working for me without any attached protein to see which one gives me less aggregation.
I checked egfp, superfoldGFP, and alph-gfp, and nowGFP,… and found that mostly the position 225 is a T. I cannot find A225. There is either T225 or A226, or even A227
May I ask you which type of GFP you are using? and what is the mutation of A225?