Organelle Marker FAQs
1. What are the fluorescent proteins used for the organelle markers?
The fluorescent proteins are turboGFP and turboRFP from Evrogen. Both proteins are super bright with brightness % compared to EGFP of 112 and 187, respectively. They are both fast maturing, allowing detection of fluorescence as early as 8-10 hrs post transfection. In spite of their dimeric structures, tGFP and tRFP have been successfully used as fusion protein for subcellular structure labeling. All OriGene's organelle markers are tested individually for correct labeling
2. What is the excitation/emission wavelength of tGFP?
The excitation/ emission max = 482/ 502 nm. tGFP can be detected using common fluorescence filter sets for EGFP, FITC, and other green dyes. Recommended Omega Optical filter sets are QMAX_Green, XF100_2, XF100_3, XF115_2, and XF116_2
3. What is the excitation/emission wavelength of RFP?
The excitation/emission max=553/574 nm. tRFP can be detected using common fluorescence filter sets for Texas Red. Recommended Omega Optical filter sets are QMAX_Yellow, XF108_2, XF101_2, and XF111_2. tRFP can also be detected using TRITC filter set.
4. Can I use a regular fluorescent microscope to obtain organelle images?
A: Conventional fluorescent microscope can be used for viewing some of the organelle or subcellular structures. However the images are usually lack the desired clarity. Confocal microscope is strongly recommended for obtaining sharp organelle images. All of OriGene's validation data was obtained using a confocal microscope.
5. What antibody can I use to detect the tGFP fusion protein on a Western blot?
We recommend the catalog# TA150041 (Mouse monoclonal turboGFP antibody)
6. What antibody can I use to detect the RFP fusion protein on a Western blot?
We recommend the catalog# TA150061 (mouse monoclonal turboRFP antibody)
7. Can I make stable mammalian cell lines with the organelle markers?
Yes. All organelle marker plasmids carry a neomycin resistance marker for G418 selection.
8. What positive control should I use with these markers?
Optional nuclear protein marker, such as cyclin D1, with additional cost. If you have an antibody specific for an organelle, you can also use that as a reference point.
9. Where can I find the sequence and cloning sites for my Organelle marker?
All organelle markers are cloned into the SgfI and MluI sites. The vector sequence are available from website at http://www.origene.com/cdna/cellPainter_Vector.aspx
10. Which vector should I use as a negative control for my GFP-tagged or RFP-tagged marker?
The corresponding empty vector can serve as good negative control. Detailed info can be found at http://www.origene.com/cdna/cellPainter_Vector.aspx
11. Can I shuttle my ORF using your PrecisionShuttle system to change my fluorescent tag?
Yes. All organelle markers can be easily shuttled among any of the Destination vectors using the PrecisonShuttle system. For further information, please see our website at http://www.origene.com/cdna/trueorf/destinationvector.aspx
12. How are the organelle marker tested?
Each organelle marker plasmids are tested by transfection into HEK293 or SKOv3 cells followed by imaging via confocal microscopy. Appropriate organelles or subcellular structures were labeled and the data can be viewed online at http://www.origene.com/assets/documents/trueorf/FP-Images.pdf
13. How should I cite your product?
Here is our guideline:
Full Product Name, OriGene Technologies Inc., Rockville, MD, USA, Catalog #, Lot #.
In addition, we would love to hear from you when the paper is published. Email us a copy of the accepted manuscript and receive a special gift.