Difference between revisions of "Part:BBa J63000:Experience"
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The fluorescence from mCherry can be readily visualized by microscopy when mCherry is expressed from a reasonable promoter, i.e. CTS1, MET25, in addition to very strong promoters, i.e. GAL1, ADH1. Because mCherry's absorption band only minimially overlaps with a 568 nm laser line, the signal to noise for FACS measurements is lower than microscopy, but the signal is clearly distinguishable from the noise. The main benefit of mCherry over other red fluorescent proteins is that it is monomeric and that it has a relatively short maturation time (15 min in vitro[1] and 45 min in vivo in yeast[2]). One caveat is that in addition to the full-length mCherry, several smaller and fainter MW bands appear on a western blot of a whole cell extract from Saccharomyces cerevisiae expressing the mCherry construct. When overexpressed in E. coli, the same construct give rise to only a single band on a western. <br> | The fluorescence from mCherry can be readily visualized by microscopy when mCherry is expressed from a reasonable promoter, i.e. CTS1, MET25, in addition to very strong promoters, i.e. GAL1, ADH1. Because mCherry's absorption band only minimially overlaps with a 568 nm laser line, the signal to noise for FACS measurements is lower than microscopy, but the signal is clearly distinguishable from the noise. The main benefit of mCherry over other red fluorescent proteins is that it is monomeric and that it has a relatively short maturation time (15 min in vitro[1] and 45 min in vivo in yeast[2]). One caveat is that in addition to the full-length mCherry, several smaller and fainter MW bands appear on a western blot of a whole cell extract from Saccharomyces cerevisiae expressing the mCherry construct. When overexpressed in E. coli, the same construct give rise to only a single band on a western. <br> | ||
| − | More information about the absolute expression levels, relationship between fluorescence and number of molecules, maturation rate, and photobleaching rate can be found in [2]. <br> | + | More information on the excitation and emission characteristics and the amino acid sequence can be found in [1]. More information about the absolute expression levels, relationship between fluorescence and number of molecules, maturation rate, and photobleaching rate can be found in [2]. <br> |
[1] Shaner, N.C., et al."Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein." Nat Biotechnol. 22(12):1567-72.<br> | [1] Shaner, N.C., et al."Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein." Nat Biotechnol. 22(12):1567-72.<br> | ||
[2] Ajo-Franklin, C. M. and Drubin, D. A. et al. "Rational design of memory in eukaryotic cells." Genes Dev., 21(18):2271-6. | [2] Ajo-Franklin, C. M. and Drubin, D. A. et al. "Rational design of memory in eukaryotic cells." Genes Dev., 21(18):2271-6. | ||
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Latest revision as of 17:51, 26 November 2007
This experience page is provided so that any user may enter their experience using this part.
Please enter
how you used this part and how it worked out.
Applications of BBa_J63000
mCherry has been used to track protein expression and protein localization as a function of time.
User Reviews
UNIQbde5073a4c4757e5-partinfo-00000000-QINU
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BBa_J63000 Caroline Ajo-Franklin |
The fluorescence from mCherry can be readily visualized by microscopy when mCherry is expressed from a reasonable promoter, i.e. CTS1, MET25, in addition to very strong promoters, i.e. GAL1, ADH1. Because mCherry's absorption band only minimially overlaps with a 568 nm laser line, the signal to noise for FACS measurements is lower than microscopy, but the signal is clearly distinguishable from the noise. The main benefit of mCherry over other red fluorescent proteins is that it is monomeric and that it has a relatively short maturation time (15 min in vitro[1] and 45 min in vivo in yeast[2]). One caveat is that in addition to the full-length mCherry, several smaller and fainter MW bands appear on a western blot of a whole cell extract from Saccharomyces cerevisiae expressing the mCherry construct. When overexpressed in E. coli, the same construct give rise to only a single band on a western. |
UNIQbde5073a4c4757e5-partinfo-00000002-QINU