Hi Hanna,
I'm guessing "FRAM" is a typo and you meant FRAP?
I'm no expert in FRAP, but it seems to me that the answer may depend
on the specific question your friend wants to address... is she
interested in tracking histone movement from cytoplasm to nucleus,
inside nucleus, or in specific regions of the nucleus?
This being said, typically FRAP measures the recovery of fluorescence
within a bleached area, from which diffusion rates or other
parameters are calculated. In this respect, the exact area that is
bleached should not matter, as long as the calculations are done
right (although the shape of the bleached are may matter for the
calculations, and the relative sizes of the bleached and unbleached
areas may have some practical importance).
Now, things may be more complicated than this, in which case your
friend is probably better off reading some methods papers before
undertaking the experiments. One example could be:
Carrero et al: Using FRAP and mathematical modeling to determine the
in vivo kinetics of nuclear proteins. Methods 29: 14-28 (2003). There
are many others..
Julio.
--
Julio Vazquez,
Fred Hutchinson Cancer Research Center
Seattle, WA
http://www.fhcrc.org/
=
On Jun 1, 2009, at 3:17 AM, Hanna Sas Nowosielska wrote:
> Dear All,
>
> My friend who works on FRAM has a question concerning a bleaching
> area. As I'm not experienced with that kind of expreiments I would
> like to ask you for advice. This is her question:
> "I'm trying to compare the dynamics of histone proteins in
> different cell types in Arabidopsis thaliana roots. Now, the
> problem is that those cells types differ with the size of the
> nuclei. The question is, what is the most correct thing to do,
> bleach always the same area (the same size) or bleach always the
> same proportion of the nucleus (lets say half of the nucleus or so)?"
>
> All best,
> Hanna
> --
> Department of Plant Anatomy and Cytology
> Faculty of Biology and Environmental Protection
> University of Silesia
> Jagiellonska Str. 28
> 40-032 Katowice
> Poland
>
>
>
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