FRAP question
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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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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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In reply to this post by Hanna_SN
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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In reply to this post by Hanna_SN
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 On Jun 1, 2009, at 3:17 AM, Hanna Sas Nowosielska wrote: Dear All, |
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In reply to this post by Hanna_SN
Dear Hanna, From what I understand from your question, your friend would
like to quantify the dynamics of the histone proteins in terms of a diffusion
coefficient. You are right to ask these questions because the recovery due to
diffusion will depend on both the size and shape of the bleach area, as well as
the shape and volume of the surrounding compartment (i.e. the nucleus). I would
recommend the following: 1. If the aim is just to perform some qualitative experiments (‘does
the recovery in one nucleus go ‘faster’ than in another one’),
photobleach an identical small region in each nucleus and only analyze the initial
part of the recovery curve. At later times the diffusion process will be
influenced by the walls of the nucleus, and as different nuclei have different
sizes and shapes, this should be avoided. So, the ‘initial stage’ means
the time it takes for the diffusion front to reach the nuclear wall (you could try
to estimate this from your recovery images). 2. If the aim is to perform quantitative experiments, you should
really resort to an appropriate FRAP model/method. For example, the method by
Carrero et al., Methods 29, 2003 would be a good place to start. Mind, however,
that in this work the resolution of the bleaching beam and imaging apparatus is
not taken into account, so the ‘strip’ they are talking about
should be quite broad for this to be valid (i.e. many times wider than the
resolution of the photobleaching beam). If you have access to a two-photon microscope,
you could have a look at Mazza et al, Biophys J 95, 2008. It has the advantage
of full compatibility with high NA lenses. Hope this helps, Best regards, Kevin Van: Confocal
Microscopy List [mailto:[hidden email]] Namens Hanna
Sas Nowosielska Dear All,
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