Re: Frap analysis

Posted by Anchall ............ on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Frap-analysis-tp591758p591761.html

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Dear Kevin,
         Thanks a lot for replying.I work with different RAS proteins
which are mostly localised in plasma membrane and some intracellular
organelles like golgi and ER.All these proteins have c terminal post
translational modifications .Now what I want to see is if different
post translational mutants of these proteins have similar or different
kinetics inside living cells.I am using MDCK cells for these expt.I
want to frap the mutants and the wt and see if there is any difference
in the diffusion kinetics and to find out if these modifications have
any role to play in defining localisation and kinetics of these
proteins.I will like to quantify the data once I get these frap
mesurements. Regarding which frap model I should use I am bit
confused.I dont know which model I should use to fit my data.
Also I didnt understand how sample geometry can help me select the
frap method .You also mentioned about checking some reference
solutions first. Can you guide me what kind of samples I should use
for reference.
Hope you will reply soon
Regards
Anchal

On Jan 25, 2008 7:47 AM, Kevin Braeckmans <[hidden email]> wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Dear Anchal,
>
> It would be helpful if you could give some more details - what are you
> trying to measure, what kind of sample are you using, do you want to obtain
> quantitative data and if so, which FRAP method would you like to use etc.
>
> Very generally, the difficulty is not the image processing, which usually is
> just averaging the fluorescence intensity in some ROI and normalizing that
> to a reference area and the fluorescence before bleaching. If you want to
> obtain quantitative data, the real challenge is to perform the experiment
> according to the limits implied in the FRAP model (which are not always
> clearly discussed in articles). Only then one can expect to obtain
> reasonable values from a best fit of the model to the recovery curve
> (obtaining a value does not necessarily mean it is a correct one).
>
> If you are starting from scratch, I would advise the following:
> 1. Define your experiment, the sample geometry being the most important
> factor (3D extended sample, sample with limited thickness, sample of limited
> volume)
> 2. Choose a FRAP method which is suitable for your experiment and which can
> be performed by your instrument.
> 3. Practice on some reference solutions of known diffusion coefficient to
> see if the method works correctly (each FRAP method has some limits which
> are implied in the mathematical derivation - make sure to perform your
> experiment accordingly)
> 4. Apply the method to your actual samples.
>
> You also might want to check out the archives since this topic has been
> discussed several times on this list.
>
> Best regards,
>
> Kevin
>
>
> Kevin Braeckmans, Ph.D.
> Lab. General Biochemistry and Physical Pharmacy
> Ghent University
> Harelbekestraat 72
> 9000 Ghent
> Belgium
> Tel: [phone]
> Fax: [phone]
> > -----Oorspronkelijk bericht-----
> > Van: Confocal Microscopy List [mailto:[hidden email]]
> > Namens Anchal Chandra
> > Verzonden: donderdag 24 januari 2008 18:31
> > Aan: [hidden email]
> > Onderwerp: Frap analysis
> >
> > Search the CONFOCAL archive at
> > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> >
> > Hello all,
> >        I am new FRAP user.CAN ANYONE HELP ME WITH ANALYSIS FRAP DATA
> > USING
> > IMAGE J AND IGOR PRO.aLSO HOW CAN i quantify my data.It will be nice if
> > someone can tell me step by step procedure to analyse frap data .I use
> > leica
> > sp5 for collecting frap data.
> > Hope to hear from you all soon
> > Thanks
> > Anchal
>