Hi Richard Unfortunately the photo was missing. Perhaps you might want to embed a link instead The paper actually discusses the binding to mitochondrial DNA, but for imaging or as an application with the LSC this should not be a problem. Several years ago I looked at "non-specific" binding of avidins and found in my case that high salt (10x physiological NaCl) seemed to significantly reduce it on skin samples which I attributed to the neutralising effect of high salt on electrostatic binding. Perhaps you could a comment on that possibility. For those interested you can have a look at the kit for measuring it by flow http://www.kamiyabiomedical.com/04LifeScienceResearchProducts/PackageInserts/3/DN001.htmor buy it a bit cheaper from your favourite supplierRegardsGerhard Nebe-von-CaronResearch ScientistApplied Science & Technology GroupSEAC - Safety and Environmental Assurance CentreUnilever Research, Colworth Laboratory, Sharnbrook, UK - MK44 1LQTel: +44 (0)1234 264822, Fax: +44 (0)1234 222552E- mailto:Gerhard.Nebe-von-Caron@unilever.com-----Original Message-----From: Richard Haugland [SMTP:richard.haugland@probes.com]Sent: Wednesday, October 03, 2001 2:53 AMTo: Gerhard Nebe-von-CaronCc: dick@probes.com; Cytometry Mailing ListSubject: Re: Any thoughts on the detection of Ionizing radiationeffects in ratsGerhard,That's an interesting paper whose significance I had missed. We have not testedthat property fo avidins. Oxidized guanosine has one ring that looks sort oflike the urea part of biotin but I am surprised that the affinity is highenough because it has the ribose off one of those nitrogen atoms and the secondaromaticring of the purine bears little resemblence to the rest of biotin.We see nice selective staining of mitochondria by avidins in total absence ofbiotinylated probes so there could be difficulty staining 8-hydroxyguanosincein cells with avidins if there is only low-level damage. It is definitely apotential problem using any avidin or streptavidin conjugate with fixed cellpreps thatfew people are aware of.[Photo: 46KB]The intermediate filaments in bovine pulmonary artery endothelial cells,localized using anti-desmin antibody (mousemonoclonal 131-15014), which was visualized with Alexa Fluor 647 goatanti–mouse IgG conjugate. Endogenousbiotin in themitochondria was labeled with an Alexa Fluor 546 streptavidin conjugate and DNA in the cell was stained withblue-fluorescent DAPI.Gerhard Nebe-von-Caron wrote:> It would be interesting to compare that with the paper of>http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=9448838&dopt=Abstractas> they claim the "Direct detection of 8-oxodeoxyguanosine and 8-oxoguaninebyavidin and> its analogues" as a measure of oxidative DNA damage. It looksverystraightforward> but suggests you should not block with streptavidin but justmeasure it's> binding.RegardsGerhard-----Original Message-----From: Richard Haugland> [SMTP:richard.haugland@probes.com]Sent: Friday, September 28, 2001> 8:20 PMTo: Cytometry Mailing ListSubject: Re: Any thoughts on the detection> of Ionizing radiationeffects in ratsThere is a possibility that the reagent> ARPhttp://www.probes.com/servlets/product?region=Select+Region&item=10550would> be useful.This biotin derivative reacts with abasic sites of nucleic acids> (nucleic acidsthat havelost their base but not the backbone structure> and thus generate aldehydes.ARP has been used to detect this event in living>cells:http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10639140&dopt=Abstractand> abasic sites produced by reactive-oxygen>specieshttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=1567824&dopt=Abstracthttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=8347625&dopt=Abstracthttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11020331&dopt=AbstractIonizing> radiation also produces abasic sites. Use of agreen-fluorescentstreptavidinconjugate> should work in flow cytometry (or imaging); however, I recommend thatyoublockendogenous> biotin first with streptavidin followed by excess biotin.Mitochondria,inparticular,> have endogenous biotinylated proteins that could interferewithlow-leveldetection. Cells> with endogenous damage by ROS may also react as positive so youwillneedcontrols.Simon> Monard wrote:> Hi> Could you look for chomosome damage by flow cytometry. Idon't> know how rarechromosome> damage would be. Its not very hard to preparechomsome preps> from rats,either con> A stimulated blood or perhaps you could look atgranukoma pouch> assay cells.Just> a thought. We made some chromosome paints for rat andtried to look> forabnormal> chomosomes by microscopy also.> Simon> FLOWers>> I amin need of> some help.>> We want to look at the protective effect of drug X on radiationinduced>> damage in the rat, by Flow Cytometry.> Does anyone have any thoughts??>> Iknow this> is potentially a very broad subject, but I would appreciate any> thoughts onpotential> avenues of approach>> > thank you for your time and help > > > > Philip Barren
This archive was generated by hypermail 2b29 : Wed Apr 03 2002 - 11:57:55 EST