I would like to examine microtubule dynamics in live cell lines in combination when co-expressed with other GFP-tagged proteins, hence I require a red chimaera.
Transfection is transient (Lipofectamine-type), not viral. I will image on a confocal using standard R/G filter sets and collect time-lapse movies.
I see that Addgene distributes a human EB1 tagged at the C-terminus with monomeric RFP (original Tsien 2002 variant).
So my questions are:
(a) Does this construct express well enough in HeLa cells?
(b) Is this 'old' RFP realistically bright and stable enough to image for 10 min (its extinction coeff is only 44k compared to 72k for mCherry; similar quantum yields)?
Any advice from those with experience?
Thanks in advance.
I will do similar experiments with mRuby (PlosOne, 2009) in the next weeks. Maybe we can stay in contact to discuss this points?
We used a 2xeGFP or a mCherry construct --> see http://www.sciencedirect.com/science/article/pii/S0960982212002564
Thanks Samantha, I'm sensing that the RFP won't be bright enough. I've requested an mCherry construct from someone.
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