Fluorescent proteins
Locked
|
Dear all,
One common question I get is "What is the best combination of fluorescent proteins to use in my live cell imaging experiment?". Based on experience my advice for a double labelling experiment would be Cerulean and mVenus, or perhaps EGFP and mCherry and for a triple labelling experiment Cerulean, mVenus and mCherry. It seems that every few months a new contender to the best FP crown appears, and I was just wondering if anyone has any 'real world' experience of some of these newer variants which would lead them to chose a different combination from the ones listed above. For example, has anyone successfully used Azurite, mTeal, Emerald, YPet, TagRFP or mKate either in isolation or in combination? Does anyone have bad experiences of these FPs? Thanks, Simon Babraham Institute, UK |
|
|
Hi Simon,
It all depends on the application and the final goals. If you are working under conditions of high overexpression, that it doesn't matter much. However, if you would be using weak promoters, create cell lines expressing a fusion protein of interest, then many things become important. For example, I would use CeFP fusion protein as a reference or as a fusion to relatively abundant proteins in the cell (e.g. actin, tubulin, histones, etc). YFP channel is very good, in relation to SNR, but I may suggest a fusion to tandem, 2x_EYFP or 2x_venusYFP (with one or all three monomeric mutations). For the red channel mCherry (or mRFP1 fused to the N- and C-terminal peptides of GFP origin), like in mCherry. If your expression vectors are of viral origin, or if you may suspect aberrant splicing artefacts in your model system, then M10L version of mCherry will solve the problem (i.e. contamination by free, unfused mCherry). Autofluorescence is a serious obstacle in low light microscopy, thus I would recommend adapting your cell cultures to OPTI-MEM media with 2-3% FCS. Many others proteins are either less soluble or di- through tetramers, may suffer from the low quantum yield, low SNR, etc. Recently, there were few papers on new FPs which sounded interesting, but nothing alike "supersonic".... mCherry, especially in its 2x tandem variant is still a "trouble-maker" (has a tendency to form aggregates as fusion to a number of proteins tested myself in vivo), and I doubt that mCherry is monomeric under conditions of overexpression - in a simple BiFC experiment where mCherry was fused to split YFP (mCherry_ny and mCherry-cy), YFP signal reconstitution is as quick_and_strong as in the positive control (CD8_ny plus CD8-cy; and CD8 is known to live as a dimer). If you have any further questions, please contact me off-line. Vitaly NCI-Frederick, 301-846-6575 -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Simon Walker Sent: Friday, July 31, 2009 10:54 AM To: [hidden email] Subject: Fluorescent proteins Dear all, One common question I get is "What is the best combination of fluorescent proteins to use in my live cell imaging experiment?". Based on experience my advice for a double labelling experiment would be Cerulean and mVenus, or perhaps EGFP and mCherry and for a triple labelling experiment Cerulean, mVenus and mCherry. It seems that every few months a new contender to the best FP crown appears, and I was just wondering if anyone has any 'real world' experience of some of these newer variants which would lead them to chose a different combination from the ones listed above. For example, has anyone successfully used Azurite, mTeal, Emerald, YPet, TagRFP or mKate either in isolation or in combination? Does anyone have bad experiences of these FPs? Thanks, Simon Babraham Institute, UK ------------------------------------------------- |
|
|
In reply to this post by simon walker (BI)
Dear all,
The question raised by Simon regarding fluorescent proteins (FP) in living cells is a highly relevant question that I believe can significantly improve the outcome your experiments. In our lab we are studying calcium signaling and we want to use a good fluorescent protein (FP) together with a calcium probe. The best calcium probes (e.g. Fluo-3, Fluo-4, Oregon Green, etc.) on the market today are excited with blue light (488nm 1p or ~850nm 2p). When using these calcium probes in living cells we like to identify the genetically modified cells by a FP that does not affect the calcium recording that much. Does anyone have experiences using Fluo-3 etc. together with FPs, such as mTomato or mCherry? What is the best combination? Thank you, Per Karolinska Institutet, Sweden -----Ursprungligt meddelande----- Från: Confocal Microscopy List [mailto:[hidden email]] För Simon Walker Skickat: den 31 juli 2009 17:54 Till: [hidden email] Ämne: Fluorescent proteins Dear all, One common question I get is "What is the best combination of fluorescent proteins to use in my live cell imaging experiment?". Based on experience my advice for a double labelling experiment would be Cerulean and mVenus, or perhaps EGFP and mCherry and for a triple labelling experiment Cerulean, mVenus and mCherry. It seems that every few months a new contender to the best FP crown appears, and I was just wondering if anyone has any 'real world' experience of some of these newer variants which would lead them to chose a different combination from the ones listed above. For example, has anyone successfully used Azurite, mTeal, Emerald, YPet, TagRFP or mKate either in isolation or in combination? Does anyone have bad experiences of these FPs? Thanks, Simon Babraham Institute, UK |
|
|
Dear Per,
We like tdTomato because it is the brightest RFP, allowing us to more easily see filled dendritic spines. Also, in our hands tdTomato is less prone to aggregation than mCherry. We co-visualize Fluo4 or Fluo5F plus tdTomato in transfected hippocampal neurons. We have not systematically compared the Ca2+ responses of tdTomato transfected cells to untransfected cells. However, we definitely observe Ca2+ signals in response to glutamate uncaging near spines in tdTomato-expressing neurons, and there is nothing obviously unusual about their properties. It is not trivial to do an exact comparison to untransfected, because it is difficult to identify Fluo4-loaded spines if the cells are not also expressing a red marker. Michael
Michael J. Schell, CIV, USU Dept. Pharmacology Uniformed Services University 4301 Jones Bridge Rd. Bethesda, MD 20814 Tel: 301-295-3249 |
«
Return to Confocal Microscopy List
|
1 view|%1 views
| Free forum by Nabble | Edit this page |