Simon, There are NUMEROUS suppliers of "TUNEL" kits. I recommend you choose one depending on your application. Many/most of them actually still use the original TUNEL format that utilizes a biotin or hapten-modified nucleotide and streptavidin-HRP, etc. Since you are posting to this site, I suppose you are more interested in flow applications, I recommend choosing a format that utilizes a direct nucleotide-fluorochrome conjugate such as fluoresceine 12-dUTP. This can be efficiently incorporated onto the 3' ends of DNA fragments by TdT in the appropriate ionic environment. Many of the complete kits contain TdT incubation buffers to run the reaction in. One thing to watch out for is whether or not another natural (unlabeled) nucleotide in included in fluorochrome conjugate. A mixture allows for a longer polymer of nucleotides to be incorporated by TdT because steric hindrance can occur if you have 2 (? or several) modified nucleotides incorporated next to each other in a row. Not all companies disclose that information. Some companies such as the one that use to be called Oncor (I think Intergin bought/rescued them) also have kits based on longer wavelength dyes; but I think some of them may require something other than the usual laser used for fluorescein, so check em out before you buy. I think Boehringer (Roach Molecular) has both original and direct formats. Promega also has a direct fluoresceine-12-dUTP version of the kit will all the buffers, solubilization reagents (proteinase K), etc. Phoenix Flow Systems has a version of TUNEL (they refer to as TUBEL; but a rose by any other name...) which incorporates BrdU that is subsequently detected by fluorescein labeled antiBrdU monoclonal antibody. They claim that Br-dUTP is more efficiently incorporated because a brighter signal is generated; but, it is unclear if the brighter signal is from the increased incorporation or the amplification achieved with having multiple fluorochromes on the antibody. I am not sure that the increase in fluorescent intensity is really worth the effort of the extra steps involved with using a secondary antibody as long as you can detect the signal in apoptotic cells. Perhaps someone else could comment on this. I would recommend using one of the direct Fluoresceine-dUTP kits. You can price shop; but I recommend buying direct rather than through a distributor. I never know how long thay have had them sitting on their shelf. About a year ago, I think there was a comparison of several companies' apoptosis products, perhaps in The Scientist, Biomedical Products, or another one of those 11" x 14" rags. After all of this, have you considered sub G0 or one of the other apoptosis markers recently discussed on this list? Ty -----Original Message----- From: Simon Monard <smonard@trudeauinstitute.org> To: cyto-inbox Date: Friday, March 10, 2000 6:06 PM Subject: TUNEL KITS > >Hi All > >What other TUNEL kits are available other than the Boehringer Mannheim kit? Which ones >work the best? > >Thanks > >Simon > >Simon Monard >FACS Lab Manager >Trudeau Institute >Saranac Lake >NY12983 > >Ph 518 891 3080 X352 > >
This archive was generated by hypermail 2b29 : Sat Mar 10 2001 - 19:31:12 EST