Apart from energy transfer consider electron / photon overflow and compensation problems. Excitation of TP3 with a HENE LASER does give you a nice TOPRO signal but should not prevent FRET in the blue laser beam if there is still an energy transferring dye combination present. Regards Gerhard Nebe-von-Caron Research Scientist Applied Science & Technology Group SEAC - Safety and Environmental Assurance Centre Unilever Colworth, Sharnbrook, Bedfordshire, UK - MK44 1LQ Tel: +44 (0)1234 264822, Fax: +44 (0)1234 222552 E- mailto:Gerhard.Nebe-von-Caron@unilever.com -----Original Message----- From: DAVID M CODER [SMTP:d_coder@msn.com] Sent: Wednesday, January 30, 2002 10:20 PM To: Cytometry Mailing List Subject: Re: 7-AAD/PE problem ----- Original Message ----- From: "Corver, W.E. (PATH)" <W.E.Corver@lumc.nl> To: cyto-inbox Sent: Tuesday, January 29, 2002 9:06 AM Subject: RE: 7-AAD/PE problem > > Dear Brian, > > [part deleted] > After fixation you can use methanol, ethanol, lysolecithin or detergents. > For nuclear staining, we obtained the best results with methanol. > Second, we also observed reduction of FITC as well as PE fluorescence using > PI and TO-PRO-3 as DNA stain. (Cytometry 15:117-128, 1994 and Cytometry > 28:329-336, 1997). The phenomenon could not be fully explained by absorption > or energy transfer. This was very limited as demonstrated by fluorometry > experiments using dye mixtures at the lab of Hans Tanke (Sylvius Lab, > Leiden). What is causing the effect is still unclear to us but maybe > somebody else has a suggestion. Energy transfer seems the best explanation. FITC and PI are a near optimal pair; PE and PI less so, but still there is overlap. PI and TO-PRO-3 are a very good pair: the emission of PI overlaps well the absorption of TO-PRO-3 (e.g., a HeNe laser at 633nm excites TP-3 very well), and both dyes intercalate into DS-DNA. In fact, the only way to observe TO-PRO-3 fluorescence when only a 488nm line is available is by transfer from PI. All such energy transfer (FITC, PE --> PI; PI-->TP3) is avoided with direct excitation of TP-3 by red (633 to 647nm) excitation. Best regards, Dave ---------------- David M. Coder, Ph.D. Consultant in Cytometry email: d_coder@msn.com tel./messages: 206-499-3446 > -----Original Message----- > From: Newsom, Brian S. [mailto:BSNEWSOM@txccc.org] > Sent: maandag 28 januari 2002 22:02 > To: cyto-inbox > Subject: 7-AAD/PE problem > > > > Question to all of you DNA experts. We have someone who is tring to run an > experiment staining a nuclear localized protien with PE and then staining > with 7-AAD. The 7-AAD staining works well with a good CV. The antibody > staining looks decent (although low percentage and fairly dim) when by > itself, but when the 7-AAD is added the antibody staining totally goes away. > Is there an energy transfer or steric hinderance issue that may be going on? > Any help appreciated. > > Brian >
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