FRAP problems
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Hi all, I would like to run a FRAP experiment using our Leica TCS SP2. We are studying the turnover of a protein involved in actin nucleation and turnover in the lamellipodia, using a GFP construct. Firstly how is it best to determine the parameters for FRAP, i.e. how do you know how many prebleach, bleach and post bleach images to capture? On previous attempts I have used 4 bleach iterations using the fly mode. I assume that you should be able to see in the viewer as the ROI bleaches? does the size of the ROI have a large impact on how long the area takes to bleach? So far I have not been able to bleach the region using our argon laser 488 line at 100% power. Should I use more than one laser line? I just find this odd as i'd expect the region to bleach quickly at 100% power output. Thanks in advance, Matt Pearson. -- Imaging Technician Cell Biology Division Institute of Ophthalmology University College London EC1V 9EL 020 7608 6857 [hidden email] |
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal MIME-Version: 1.0 Content-Type: multipart/alternative; boundary="0-112221368-1216647116=:81787" --0-112221368-1216647116=:81787 Content-Type: text/plain; charset=iso-8859-1 Content-Transfer-Encoding: quoted-printable Hi=A0all!=0AI have the same problem like Matt Pearson. Different than his p= roblem, the=A0protein that I want to bleach is=A0CFP tagged fusion protein = and i use carl zeiss LSM 510.=A0 I try to do frap experiments since last on= e month and I still couldnt standardize my experiments. Efficiency of bleac= hing is very low -around 30%.=A0I will also like to hear your suggestions t= oo. (a paper on=A0troubleshooting etc.=A0may be helpful)=A0=0AHave a good d= ay,=0ASerra Akinturk=0A=A0=0A=A0=0A=0A=0A=0A----- Original Message ----=0AF= rom: Matthew Pearson <[hidden email]>=0ATo: [hidden email]= FFALO.EDU=0ASent: Monday, July 21, 2008 1:39:25 PM=0ASubject: FRAP problems= =0A=0ASearch the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-b= in/wa?S1=3Dconfocal =0AHi all,=0A=0AI would like to run a FRAP experiment u= sing our Leica TCS SP2.=A0 We are studying the turnover of a protein involv= ed in actin nucleation and turnover in the lamellipodia, using a GFP constr= uct.=A0 Firstly how is it best to determine the parameters for FRAP, i.e. h= ow do you know how many prebleach, bleach and post bleach images to capture= ?=A0 On previous attempts I have used 4 bleach iterations using the fly mod= e.=A0 I assume that you should be able to see in the viewer as the ROI blea= ches?=A0 does the size of the ROI have a large impact on how long the area = takes to bleach?=A0 So far I have not been able to bleach the region using = our argon laser 488 line at 100% power.=A0 Should I use more than one laser= line?=A0 I just find this odd as i'd expect the region to bleach quickly a= t 100% power output.=0A=0AThanks in advance,=0A=0AMatt Pearson.=0A=0A=0A=0A= =0A=0A-- =0AImaging Technician=0ACell Biology Division=0AInstitute of Ophth= almology=0AUniversity College London=0AEC1V 9EL=0A020 7608 6857=0Amatthew.p= [hidden email]=0A=0A=0A --0-112221368-1216647116=:81787 Content-Type: text/html; charset=us-ascii <html><head><style type="text/css"><!-- DIV {margin:0px;} --></style></head><body><div style="font-family:arial, helvetica, sans-serif;font-size:14pt"><DIV></DIV> <DIV>Hi all!</DIV> <DIV> </DIV> <DIV>I have the same problem like Matt Pearson. Different than his problem, the protein that I want to bleach is CFP tagged fusion protein and i use carl zeiss LSM 510. I try to do frap experiments since last one month and I still couldnt standardize my experiments. Efficiency of bleaching is very low -around 30%. I will also like to hear your suggestions too. (a paper on troubleshooting etc. may be helpful) </DIV> <DIV> </DIV> <DIV>Have a good day,</DIV> <DIV> </DIV> <DIV>Serra Akinturk<BR> </DIV> <DIV><FONT face="verdana, helvetica, sans-serif"> <P class=MsoNormal style="MARGIN: 0cm 0cm 0pt"><SPAN style="FONT-SIZE: 11pt"><FONT face="garamond, new york, times, serif"><FONT size=3></FONT></FONT></SPAN></P><SPAN style="FONT-SIZE: 11pt"><FONT face="garamond, new york, times, serif"><FONT size=3><SPAN style="COLOR: black"></SPAN></FONT></FONT></SPAN> </DIV></FONT> <DIV><BR></DIV> <DIV style="FONT-SIZE: 14pt; FONT-FAMILY: arial, helvetica, sans-serif"><BR> <DIV style="FONT-SIZE: 12pt; FONT-FAMILY: times new roman, new york, times, serif">----- Original Message ----<BR>From: Matthew Pearson <[hidden email]><BR>To: [hidden email]<BR>Sent: Monday, July 21, 2008 1:39:25 PM<BR>Subject: FRAP problems<BR><BR>Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal <BR>Hi all,<BR><BR>I would like to run a FRAP experiment using our Leica TCS SP2. We are studying the turnover of a protein involved in actin nucleation and turnover in the lamellipodia, using a GFP construct. Firstly how is it best to determine the parameters for FRAP, i.e. how do you know how many prebleach, bleach and post bleach images to capture? On previous attempts I have used 4 bleach iterations using the fly mode. I assume that you should be able to see in the viewer as the ROI bleaches? does the size of the ROI have a large impact on how long the area takes to bleach? So far I have not been able to bleach the region using our argon laser 488 line at 100% power. Should I use more than one laser line? I just find this odd as i'd expect the region to bleach quickly at 100% power output.<BR><BR>Thanks in advance,<BR><BR>Matt Pearson.<BR> <DIV class=moz-signature><BR> <DIV class=moz-signature> <DIV class=moz-signature><BR><PRE class=moz-signature><FONT color=#3366ff><FONT color=#3333ff><FONT face=Arial>-- Imaging Technician Cell Biology Division Institute of Ophthalmology University College London EC1V 9EL 020 7608 6857 <A class=moz-txt-link-abbreviated href="mailto:[hidden email]" target=_blank rel=nofollow ymailto="mailto:[hidden email]">[hidden email]</A></FONT></FONT><FONT color=#3333ff> </FONT> </FONT></PRE></DIV></DIV></DIV></DIV></DIV></div><br> </body></html> --0-112221368-1216647116=:81787-- |
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In reply to this post by Matthew Pearson-3
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= Dear Matt and Serra, FRAP is an advanced technique, and it is probably best to have a look at some relevant publications. Below, I list a few papers (subscription may be needed to view) with protocols and general guidelines, and there are probably many more in the Methods in Enzymology and similar series. What conditions will work for you depend on your specific system. I would suspect a typical Argon laser at full power will bleach most samples rather quickly on a confocal. For live imaging, we use our 488 laser at about 2-3 % max power, and we still will get some bleaching after a while. At 10% max power, we are probably working near saturation, and bleaching is already rather severe. This is something you need to determine with your specific instrument and samples. What you need to take into account is whether your protein is tethered or freely diffusible. If your protein is freely diffusible, it may take only a few seconds for fluorescence in any bleached area to recover du to diffusion. In that situation, if you need to do measurements in a small region inside the cell, you need to work really fast. Otherwise, you need to bleach all the cellular pool of protein (in the entire cell volume... make sure you are not just bleaching one focal plane). If your protein is tethered, then you can afford to work more slowly. Also, I would suggest you find and use the lowest laser power settings you can work with. Again, at full power with a 488, you may be doing some serious damage to your cells, besides bleaching your reporter protein. Julio. Carrero et al. Using FRAP and mathematical modeling to determine the in vivo kinetics of nuclear proteins. Methods 29: 14-28 (2003). in Current protocols in cell biology, 2003, supplement 18: papers by the Misteli (13.5.1-13.5.18), Lippincott-Schwartz (21.1.1-21.1.24), and Zichu (21.2.1-21.2.16) labs. -- Julio Vazquez Fred Hutchinson Cancer Research Center Seattle, WA 98109-1024 On Jul 21, 2008, at 4:39 AM, Matthew Pearson wrote: Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal |
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear colleagues it is available for free: Mazza D, Braeckmans K, Cella F, Testa I, Vercauteren D, Demeester J, De Smedt SS, Diaspro A. A New FRAP/FRAPa Method For 3-D Diffusion Measurements Based On Multi-photon Excitation Microscopy. Biophys J. 2008. http://www.biophysj.org All the best Alby ---------------------------------------------------- "Water slowly flowed under the sky" (Cesare Pavese) ----------------------------------------------------- Alberto Diaspro, EBSA President-Elect, MicroScoBIO LAMBS-IFOM, Department of Physics, University of Genoa, Via Dodecaneso 33, 16146 Genoa, Italy - fax +39-010314218 - tel +39 0103536426/309; URLs: www.lambs.it ; EBSA is Biophysics in Europe - European Biophysical Societies' Association www.ebsa.org ---------------------------------------------- |
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In reply to this post by Julio Vazquez
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear colleagues it is available for free download a FRAP paper by Mazza and colleguaes. Please download it at http://www.biophysj.org. It is related to a companion paper published on Appl opt by Mazza et al., again. All the best Alby ---------------------------------------------------- "Water slowly flowed under the sky" (Cesare Pavese) ----------------------------------------------------- Alberto Diaspro, EBSA President-Elect, MicroScoBIO LAMBS-IFOM, Department of Physics, University of Genoa, Via Dodecaneso 33, 16146 Genoa, Italy - fax +39-010314218 - tel +39 0103536426/309; URLs: www.lambs.it ; EBSA is Biophysics in Europe - European Biophysical Societies' Association www.ebsa.org ---------------------------------------------- |
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