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To All,
Just to add a comment to Jim's query, it is not just proteins/nucleic
acids that are likely to be affected by reactive species, but
unsaturated lipid side chains can form carbon centered radicals that
propagate crosslinking of the side chains in membrane bilayers. Once
the process starts, it is auto-catalytic. This is fine when linseed
oil coating a piece of wood but not so good for bilayers. In fact,
one might create domains that contain nanoscopic holes, as well as,
rigid domains with very different diffusion constants from the native
membrane. I would be interested in any references that have made a
study of these factors.
Mario
>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>>Dear Kevin,
>>
>>In principle I'm doing 2d measurements on a zeiss Lsm 510 confocal
>>scanning microscope. I have been doing spot bleaching since I have
>>started, for that seemed the easiest way to start up the experiments.
>>
>>The half time of recovery for my proteins i about one minut and I
>>tend to measure for 10 minutes so that recovery seems to be at 95 %
>>or more. Of course i have been correcting for the bleaching due to
>>the scanning durign the recovery phase, but I did not check yet if I
>>take into account the 15 percent rule that it will get better.
>>
>>I have recent evidence that the mobile and immobile fraction might
>>be heterogenous due to the fact that my protein of interest is DNA
>>binding and transcription rate influences its mobility. Inside a
>>nucleus the transcription rate is for sure heterogenous and this
>>might explain why I find huge differences.
>>
>>You say that multiple FRAP formulas exist, I would be very
>>interested in hearing about the different patterns that might be
>>used and which formulas belong to them
>>
>>Thanks for the answers
>>
>>Joost Willemse Msc.
>>WUR Dept. Molecular Biology
>>Dreienlaan 3
>>6703 HA Wageningen
>>++(31)317484574
>
>
>I'm not a FRAP'er but...
>
>I must say that it worries me that so many people who do FRAP seem to
>assume that the only thing that happens in the exposed region is that
>the dye bleaches. Starting with this assumption people then
>calculate a lot of diffusion rates.
>
>I think that this assumption is unlikely to be generally true. At the
>very least, the bleached area will have a higher concentration of the
>active oxygen intermediates that we seem to believe are a concomitant
>of the bleaching reaction.
>
>I only butt into this conversation to make sure that everyone is
>aware of Richard McIntosh's work in U. Colorado, Boulder in which he
>made mitotic MTs fluorescent, bleached a band across them and then
>looked at the same exact cells in the electron microscope. He found
>that the MTs had been destroyed in the bleached band. (Vigers et all,
>1988, JCB 207:1011). He earlier had shown that fluorescent MTs would
>only move around on a dynein-coated coverslip UNTIL they have been
>viewed at a wavelength that excited the dye. Once the dye had been
>excited, even briefly, the MTs no longer moved. (McIntosh chapter in
>a book called Motility in Cell Function edited by Frank Pepe, Joe
>Sanger and Vivian Nocknius (sp?), Academic Press, 1979 (?))
>
>Surely, such destruction might be expected to change diffusion rates?
>
>What precautions are taken to detect or guard against this problem?
>
>Cheers,
>
>Jim P.
>
>--
> ****************************************
>Prof. James B. Pawley, Ph. 608-263-3147
>Room 223, Zoology Research Building, FAX 608-265-5315
>1117 Johnson Ave., Madison, WI, 53706 [log in to unmask]
>"A scientist is not one who can answer questions but one who can
>question answers." Theodore Schick Jr., Skeptical Enquirer, 21-2:39
--
_________________________________________________________________
Mario M. Moronne, Ph.D.
NanoMed Technologies LLC
President and CTO
ph (510) 528-2400
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