(Red Fluorescent Protein)

The new red fluorescent protein, DsRed, from Clontech can be viewed, and imaged, with a variety of filter sets due to its broad absorption and emission spectra. Using HeLa cells transfected with the DsRed protein, kindly provided by Clontech, we obtained excellent images with either the HQ TRITC/Cy3 ( 41002c or 41002b) cubes or the HQ Texas Red set (41004). We also found that a custom set we have made for several researchers worked very well, as did the Phycoerythrin set (41003).

We Recommend Chroma HQ Sets:

41002c (hq545/30x, q570lp, hq620/60m)
41002b (hq545/30x, q570lp, hq610/75m)
41004 (hq560/55x, q595lp, hq645/75m)
also:
41003 (hq546/12x, q560lp, hq585/40m)
41035 (hq546/12x, q560lp, hq605/75m).

Due to the very wide absorption spectra, this protein may exhibit slightly different characteristics as it is expressed in different cells and varying condition.

We found that in our samples of DsRed transfected cells they all looked somewhat brighter with the HQ TRITC sets, while the HQ Texas RedTM (41004) gave slightly better contrast. The red-shifted TRITC (41002c), the Texas RedTM, and the custom set (41035) gave a redder signal, compared to the yellowish image of the TRITC and Phycoerythrin cubes (41002b and 41003). It did take longer to acquire the Texas RedTM images in some cells, indicating that the signal was indeed decreased. In comparing the saved images, however, the two were nearly identical in gray level. In two of the three samples that we tested the Phycoerythrin (41003) and the custom cube (41035) gave slightly brighter signals, but it is apparent that this will vary some from cell to cell and from protocol to protocol.

Furthermore, there was excellent separation between the enhanced green fluorescent protein (EGFP) and the DsRed using either the HQ TRITC filter sets or the HQ Texas RedTM filter set. There was also, virtually no yellow gfp (EYFP) visible in any of the above cubes used for DsRed.

In using dual sets, again both the FITC/TRITC dual (51004 v2) and the FITC/Texas RedTM (51006) gave very clean separation between the EGFP and the DsRed cells. The FITC/TRITC dual gave the DsRed a more yellow color, while the FITC/Texas RedTM red appeared much more red with slightly better contrast. However, due to several requests from researchers with minimal expression levels we have designed a dual set specifically for EGFP and DsRed (51019). This dual set has been tested in several labs, with excellent results.

We suspect that while both HQ TRITC and HQ Texas RedTM sets will work well for imaging the new DsRed, those situations using a second/shorter wavelength label/protein may benefit from the additional spectral separation of the HQ Texas RedTM set. This must be balanced with the slightly longer exposure time in some samples. In those cases where speed of acquisition is more important, the HQ TRITC sets will be superior.

In some cases the Phycoerythrin (41003) and custom set (41035) have a slight advantage, and produce a brighter image.

While the broad, blue-shifted absorption of the DsRed can present some difficulties, it also has its advantages. Those researchers with a 488 laser line should be able to image the enhanced green fluorescent protein (EGFP), the yellow protein (EYFP), and the red protein (DsRed) all at the same time with this single excitation...

However, due to this absorption peak it appears that using the green (EGFP) or yellow (EYFP) as a donor in FRET experiments with the DsRed as acceptor will not be possible. We are anxiously awaiting test results from several labs concerning the possibilities of using cyan (ECFP) as the donor. We will gladly pass along those results as we collect them.

Please contact us with any/all questions.

This application note written by
Michael Stanley , Ph.D.
Senior Application Scientist, Chroma Technology Corp.