Proximity Ligation Assay
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** I am trying to help a student from another lab who is trying to do proximity ligation assays to look for protein interactions. Anyone have experience with this technique? It is always hard to tell from the literature if an approach like that is robust or has a high failure rate or is extremely tricky to get to work. Thanks- Dave Dr. David Knecht Professor Department of Molecular and Cell Biology University of Connecticut 91 N. Eagleville Rd. Storrs, CT 06269 860-486-2200 |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hello David, I have a lot of experience with the Duolink PLA. And yes, the signal is robust with good antibodies, can be tricky as well- like every immunostaining procedure. Tissues sometimes give higher background and washing steps are critical. Happy to answer specific questions or discuss how to improve a protocol. Monika Monika Beck Gooz, MD, PhD Research Associate Professor Department of Drug Discovery & Biomedical Sciences Manager, Cell and Molecular Imaging Core Hollings Cancer Center Office: Drug Discovery Building DD507 Medical University of South Carolina 70 President Street, MSC 139 Charleston, SC 29425 E-mail: [hidden email] Tel: (843) 876-2363 -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Knecht, David Sent: Wednesday, October 19, 2016 9:29 AM To: [hidden email] Subject: Proximity Ligation Assay ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** I am trying to help a student from another lab who is trying to do proximity ligation assays to look for protein interactions. Anyone have experience with this technique? It is always hard to tell from the literature if an approach like that is robust or has a high failure rate or is extremely tricky to get to work. Thanks- Dave Dr. David Knecht Professor Department of Molecular and Cell Biology University of Connecticut 91 N. Eagleville Rd. Storrs, CT 06269 860-486-2200 ------------------------------------------------------------------------- This message was secured via TLS by MUSC. |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi Dave, I've also learned that it is important to image very soon after the procedure because the signal degrades rather quickly with time. Same-day imaging is ideal. Sara Sara Cole, PhD Assistant Director Senior Microscopist Campus Microscopy and Imaging Facility (CMIF) 277 Biomedical Research Tower, 460 West 12th Ave., Columbus, OH 43210 (614) 292-2814 Office / (614) 688-5564 Lab / (614) 292-4628 Fax [hidden email] cmif.osu.edu -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Gooz, Monika Sent: Wednesday, October 19, 2016 2:49 PM To: [hidden email] Subject: Re: Proximity Ligation Assay ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hello David, I have a lot of experience with the Duolink PLA. And yes, the signal is robust with good antibodies, can be tricky as well- like every immunostaining procedure. Tissues sometimes give higher background and washing steps are critical. Happy to answer specific questions or discuss how to improve a protocol. Monika Monika Beck Gooz, MD, PhD Research Associate Professor Department of Drug Discovery & Biomedical Sciences Manager, Cell and Molecular Imaging Core Hollings Cancer Center Office: Drug Discovery Building DD507 Medical University of South Carolina 70 President Street, MSC 139 Charleston, SC 29425 E-mail: [hidden email] Tel: (843) 876-2363 -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Knecht, David Sent: Wednesday, October 19, 2016 9:29 AM To: [hidden email] Subject: Proximity Ligation Assay ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** I am trying to help a student from another lab who is trying to do proximity ligation assays to look for protein interactions. Anyone have experience with this technique? It is always hard to tell from the literature if an approach like that is robust or has a high failure rate or is extremely tricky to get to work. Thanks- Dave Dr. David Knecht Professor Department of Molecular and Cell Biology University of Connecticut 91 N. Eagleville Rd. Storrs, CT 06269 860-486-2200 ------------------------------------------------------------------------- This message was secured via TLS by MUSC. |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** One of my key questions is people’s experience with how good of a technique this is for detecting protein-protein interaction, which I presume is its general application. Would one use it instead of FRET-FLIM or other co-localization techniques or in addition to? Is it more/less challenging? Does it give the same answers in general? Thanks- Dave Dr. David Knecht Professor Department of Molecular and Cell Biology University of Connecticut 91 N. Eagleville Rd. Storrs, CT 06269 860-486-2200 > On Oct 20, 2016, at 8:37 AM, Cole, Sara <[hidden email]> wrote: > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Hi Dave, > > I've also learned that it is important to image very soon after the procedure because the signal degrades rather quickly with time. Same-day imaging is ideal. > > Sara > > > Sara Cole, PhD > Assistant Director > Senior Microscopist > Campus Microscopy and Imaging Facility (CMIF) > 277 Biomedical Research Tower, 460 West 12th Ave., Columbus, OH 43210 > (614) 292-2814 Office / (614) 688-5564 Lab / (614) 292-4628 Fax > [hidden email] cmif.osu.edu > > > > -----Original Message----- > From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Gooz, Monika > Sent: Wednesday, October 19, 2016 2:49 PM > To: [hidden email] > Subject: Re: Proximity Ligation Assay > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Hello David, > > I have a lot of experience with the Duolink PLA. And yes, the signal is robust with good antibodies, can be tricky as well- like every immunostaining procedure. Tissues sometimes give higher background and washing steps are critical. Happy to answer specific questions or discuss how to improve a protocol. > > Monika > > Monika Beck Gooz, MD, PhD > Research Associate Professor > Department of Drug Discovery & Biomedical Sciences Manager, Cell and Molecular Imaging Core Hollings Cancer Center > Office: Drug Discovery Building DD507 > Medical University of South Carolina > 70 President Street, MSC 139 > Charleston, SC 29425 > E-mail: [hidden email] > Tel: (843) 876-2363 > > > > -----Original Message----- > From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Knecht, David > Sent: Wednesday, October 19, 2016 9:29 AM > To: [hidden email] > Subject: Proximity Ligation Assay > > > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > I am trying to help a student from another lab who is trying to do proximity ligation assays to look for protein interactions. Anyone have experience with this technique? It is always hard to tell from the literature if an approach like that is robust or has a high failure rate or is extremely tricky to get to work. Thanks- Dave > > Dr. David Knecht > Professor > Department of Molecular and Cell Biology University of Connecticut > 91 N. Eagleville Rd. > Storrs, CT 06269 > 860-486-2200 > > > > > > ------------------------------------------------------------------------- > This message was secured via TLS by MUSC. |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Dr. Knecht, Yes, it would be an alternate or additional method to Fret. In my experience with proximity labeling, it tends to be a bit more challenging but in general will give similar answers to fret. Since the proximity is a fixed distance/range, you might find discrepancies between less sensitive co-localization techniques and this method's results. Cheers, w -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Knecht, David Sent: Thursday, October 20, 2016 9:58 AM To: [hidden email] Subject: Re: Proximity Ligation Assay ***** To join, leave or search the confocal microscopy listserv, go to: https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=01%7C01%7Cwilliam.bodeen%40STJUDE.ORG%7Cd58c7a0b762f474687b608d3f8fa07bf%7C22340fa892264871b677d3b3e377af72%7C0&sdata=kBb4UoTiRsoyscR9bthouQlEW3egvezS%2F5yDjIQ8%2BA8%3D&reserved=0 Post images on https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&data=01%7C01%7Cwilliam.bodeen%40STJUDE.ORG%7Cd58c7a0b762f474687b608d3f8fa07bf%7C22340fa892264871b677d3b3e377af72%7C0&sdata=PfSYeOjisucecK%2FiakXnQlDmdj1XIHRulvj1KGz7z44%3D&reserved=0 and include the link in your posting. ***** One of my key questions is people’s experience with how good of a technique this is for detecting protein-protein interaction, which I presume is its general application. Would one use it instead of FRET-FLIM or other co-localization techniques or in addition to? Is it more/less challenging? Does it give the same answers in general? Thanks- Dave Dr. David Knecht Professor Department of Molecular and Cell Biology University of Connecticut 91 N. Eagleville Rd. Storrs, CT 06269 860-486-2200 > On Oct 20, 2016, at 8:37 AM, Cole, Sara <[hidden email]> wrote: > > ***** > To join, leave or search the confocal microscopy listserv, go to: > https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists. > umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=01%7C01%7Cwillia > m.bodeen%40STJUDE.ORG%7Cd58c7a0b762f474687b608d3f8fa07bf%7C22340fa8922 > 64871b677d3b3e377af72%7C0&sdata=kBb4UoTiRsoyscR9bthouQlEW3egvezS%2F5yD > jIQ8%2BA8%3D&reserved=0 Post images on > https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&data=01%7C01%7Cwilliam.bodeen%40STJUDE.ORG%7Cd58c7a0b762f474687b608d3f8fa07bf%7C22340fa892264871b677d3b3e377af72%7C0&sdata=PfSYeOjisucecK%2FiakXnQlDmdj1XIHRulvj1KGz7z44%3D&reserved=0 and include the link in your posting. > ***** > > Hi Dave, > > I've also learned that it is important to image very soon after the procedure because the signal degrades rather quickly with time. Same-day imaging is ideal. > > Sara > > > Sara Cole, PhD > Assistant Director > Senior Microscopist > Campus Microscopy and Imaging Facility (CMIF) > 277 Biomedical Research Tower, 460 West 12th Ave., Columbus, OH 43210 > (614) 292-2814 Office / (614) 688-5564 Lab / (614) 292-4628 Fax > [hidden email] cmif.osu.edu > > > > -----Original Message----- > From: Confocal Microscopy List > [mailto:[hidden email]] On Behalf Of Gooz, Monika > Sent: Wednesday, October 19, 2016 2:49 PM > To: [hidden email] > Subject: Re: Proximity Ligation Assay > > ***** > To join, leave or search the confocal microscopy listserv, go to: > https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists. > umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=01%7C01%7Cwillia > m.bodeen%40STJUDE.ORG%7Cd58c7a0b762f474687b608d3f8fa07bf%7C22340fa8922 > 64871b677d3b3e377af72%7C0&sdata=kBb4UoTiRsoyscR9bthouQlEW3egvezS%2F5yD > jIQ8%2BA8%3D&reserved=0 Post images on > https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&data=01%7C01%7Cwilliam.bodeen%40STJUDE.ORG%7Cd58c7a0b762f474687b608d3f8fa07bf%7C22340fa892264871b677d3b3e377af72%7C0&sdata=PfSYeOjisucecK%2FiakXnQlDmdj1XIHRulvj1KGz7z44%3D&reserved=0 and include the link in your posting. > ***** > > Hello David, > > I have a lot of experience with the Duolink PLA. And yes, the signal is robust with good antibodies, can be tricky as well- like every immunostaining procedure. Tissues sometimes give higher background and washing steps are critical. Happy to answer specific questions or discuss how to improve a protocol. > > Monika > > Monika Beck Gooz, MD, PhD > Research Associate Professor > Department of Drug Discovery & Biomedical Sciences Manager, Cell and > Molecular Imaging Core Hollings Cancer Center > Office: Drug Discovery Building DD507 > Medical University of South Carolina > 70 President Street, MSC 139 > Charleston, SC 29425 > E-mail: [hidden email] > Tel: (843) 876-2363 > > > > -----Original Message----- > From: Confocal Microscopy List > [mailto:[hidden email]] On Behalf Of Knecht, David > Sent: Wednesday, October 19, 2016 9:29 AM > To: [hidden email] > Subject: Proximity Ligation Assay > > > > ***** > To join, leave or search the confocal microscopy listserv, go to: > https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists. > umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=01%7C01%7Cwillia > m.bodeen%40STJUDE.ORG%7Cd58c7a0b762f474687b608d3f8fa07bf%7C22340fa8922 > 64871b677d3b3e377af72%7C0&sdata=kBb4UoTiRsoyscR9bthouQlEW3egvezS%2F5yD > jIQ8%2BA8%3D&reserved=0 Post images on > https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&data=01%7C01%7Cwilliam.bodeen%40STJUDE.ORG%7Cd58c7a0b762f474687b608d3f8fa07bf%7C22340fa892264871b677d3b3e377af72%7C0&sdata=PfSYeOjisucecK%2FiakXnQlDmdj1XIHRulvj1KGz7z44%3D&reserved=0 and include the link in your posting. > ***** > > I am trying to help a student from another lab who is trying to do > proximity ligation assays to look for protein interactions. Anyone > have experience with this technique? It is always hard to tell from > the literature if an approach like that is robust or has a high > failure rate or is extremely tricky to get to work. Thanks- Dave > > Dr. David Knecht > Professor > Department of Molecular and Cell Biology University of Connecticut > 91 N. Eagleville Rd. > Storrs, CT 06269 > 860-486-2200 > > > > > > ---------------------------------------------------------------------- > --- This message was secured via TLS by MUSC. ________________________________ Email Disclaimer: www.stjude.org/emaildisclaimer Consultation Disclaimer: www.stjude.org/consultationdisclaimer |
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In reply to this post by Knecht, David
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi David, I did co-localization studies parallel to PLA to prove that they show similar result. I also did FRET and found that PLA is less challenging than FRET. One very important difference between PLA and FRET is that with FRET you have to overexpress proteins that may or may not fold/transport/localize in the cells the way the naturally occurring ones. PLA can show interaction between endogenous proteins and that is a big advantage. Monika Monika Beck Gooz, MD, PhD Research Associate Professor Department of Drug Discovery & Biomedical Sciences Manager, Cell and Molecular Imaging Core Hollings Cancer Center Office: Drug Discovery Building DD507 Medical University of South Carolina 70 President Street, MSC 139 Charleston, SC 29425 E-mail: [hidden email] Tel: (843) 876-2363 Reminder: Please acknowledge your use of the Cell & Molecular Imaging Shared Resource in your publications. Suggested language is as follows: “Supported in part by the Cell & Molecular Imaging Shared Resource, Hollings Cancer Center, Medical University of South Carolina (P30 CA138313) and the Shared Instrumentation Grant S10 OD018113”. Thank you. -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Knecht, David Sent: Thursday, October 20, 2016 10:58 AM To: [hidden email] Subject: Re: Proximity Ligation Assay ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** One of my key questions is people’s experience with how good of a technique this is for detecting protein-protein interaction, which I presume is its general application. Would one use it instead of FRET-FLIM or other co-localization techniques or in addition to? Is it more/less challenging? Does it give the same answers in general? Thanks- Dave Dr. David Knecht Professor Department of Molecular and Cell Biology University of Connecticut 91 N. Eagleville Rd. Storrs, CT 06269 860-486-2200 > On Oct 20, 2016, at 8:37 AM, Cole, Sara <[hidden email]> wrote: > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Hi Dave, > > I've also learned that it is important to image very soon after the procedure because the signal degrades rather quickly with time. Same-day imaging is ideal. > > Sara > > > Sara Cole, PhD > Assistant Director > Senior Microscopist > Campus Microscopy and Imaging Facility (CMIF) > 277 Biomedical Research Tower, 460 West 12th Ave., Columbus, OH 43210 > (614) 292-2814 Office / (614) 688-5564 Lab / (614) 292-4628 Fax > [hidden email] cmif.osu.edu > > > > -----Original Message----- > From: Confocal Microscopy List > [mailto:[hidden email]] On Behalf Of Gooz, Monika > Sent: Wednesday, October 19, 2016 2:49 PM > To: [hidden email] > Subject: Re: Proximity Ligation Assay > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Hello David, > > I have a lot of experience with the Duolink PLA. And yes, the signal is robust with good antibodies, can be tricky as well- like every immunostaining procedure. Tissues sometimes give higher background and washing steps are critical. Happy to answer specific questions or discuss how to improve a protocol. > > Monika > > Monika Beck Gooz, MD, PhD > Research Associate Professor > Department of Drug Discovery & Biomedical Sciences Manager, Cell and > Molecular Imaging Core Hollings Cancer Center > Office: Drug Discovery Building DD507 > Medical University of South Carolina > 70 President Street, MSC 139 > Charleston, SC 29425 > E-mail: [hidden email] > Tel: (843) 876-2363 > > > > -----Original Message----- > From: Confocal Microscopy List > [mailto:[hidden email]] On Behalf Of Knecht, David > Sent: Wednesday, October 19, 2016 9:29 AM > To: [hidden email] > Subject: Proximity Ligation Assay > > > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > I am trying to help a student from another lab who is trying to do > proximity ligation assays to look for protein interactions. Anyone > have experience with this technique? It is always hard to tell from > the literature if an approach like that is robust or has a high > failure rate or is extremely tricky to get to work. Thanks- Dave > > Dr. David Knecht > Professor > Department of Molecular and Cell Biology University of Connecticut > 91 N. Eagleville Rd. > Storrs, CT 06269 > 860-486-2200 > > > > > > ---------------------------------------------------------------------- > --- This message was secured via TLS by MUSC. ------------------------------------------------------------------------- This message was secured via TLS by MUSC. |
 
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Sylvie Le Guyader |
Re: Proximity Ligation Assay
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Same experience here.
We find PLA easier than fret. PLA has a much better SNR and as said. you do not need overexpression. The disadvantage is that you get an on/off answer, not a gradual answer where the molecular distance (between the fluorophores) may be deducted. Additionally, crap in, crap out. With a poor antibody, you get pour PLA. Med vänlig hälsning / Best regards Sylvie @@@@@@@@@@@@@@@@@@@@@@@@ Sylvie Le Guyader, PhD Live Cell Imaging Facility Manager Karolinska Institutet- Bionut Hälsovägen 7, Novum, G lift, floor 6 <a href="tel:14157">14157 Huddinge Sweden mobile: <a href="tel:+46 (0) 73 733 5008">+46 (0) 73 733 5008 office: <a href="tel:+46 (0) 08-524 811 72">+46 (0) 08-524 811 72 LCI website ---- Gooz, Monika wrote ---- *****
To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi David, I did co-localization studies parallel to PLA to prove that they show similar result. I also did FRET and found that PLA is less challenging than FRET. One very important difference between PLA and FRET is that with FRET you have to overexpress proteins that may or may not fold/transport/localize in the cells the way the naturally occurring ones. PLA can show interaction between endogenous proteins and that is a big advantage. Monika Monika Beck Gooz, MD, PhD Research Associate Professor Department of Drug Discovery & Biomedical Sciences Manager, Cell and Molecular Imaging Core Hollings Cancer Center Office: Drug Discovery Building DD507 Medical University of South Carolina 70 President Street, MSC 139 Charleston, SC 29425 E-mail: [hidden email] Tel: (843) 876-2363 Reminder: Please acknowledge your use of the Cell & Molecular Imaging Shared Resource in your publications. Suggested language is as follows: “Supported in part by the Cell & Molecular Imaging Shared Resource, Hollings Cancer Center, Medical University of South Carolina (P30 CA138313) and the Shared Instrumentation Grant S10 OD018113”. Thank you. -----Original Message----- From: Confocal Microscopy List [[hidden email]] On Behalf Of Knecht, David Sent: Thursday, October 20, 2016 10:58 AM To: [hidden email] Subject: Re: Proximity Ligation Assay ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** One of my key questions is people’s experience with how good of a technique this is for detecting protein-protein interaction, which I presume is its general application. Would one use it instead of FRET-FLIM or other co-localization techniques or in addition to? Is it more/less challenging? Does it give the same answers in general? Thanks- Dave Dr. David Knecht Professor Department of Molecular and Cell Biology University of Connecticut 91 N. Eagleville Rd. Storrs, CT 06269 860-486-2200 > On Oct 20, 2016, at 8:37 AM, Cole, Sara <[hidden email]> wrote: > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Hi Dave, > > I've also learned that it is important to image very soon after the procedure because the signal degrades rather quickly with time. Same-day imaging is ideal. > > Sara > > > Sara Cole, PhD > Assistant Director > Senior Microscopist > Campus Microscopy and Imaging Facility (CMIF) > 277 Biomedical Research Tower, 460 West 12th Ave., Columbus, OH 43210 > (614) 292-2814 Office / (614) 688-5564 Lab / (614) 292-4628 Fax > [hidden email] cmif.osu.edu > > > > -----Original Message----- > From: Confocal Microscopy List > [[hidden email]] On Behalf Of Gooz, Monika > Sent: Wednesday, October 19, 2016 2:49 PM > To: [hidden email] > Subject: Re: Proximity Ligation Assay > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Hello David, > > I have a lot of experience with the Duolink PLA. And yes, the signal is robust with good antibodies, can be tricky as well- like every immunostaining procedure. Tissues sometimes give higher background and washing steps are critical. Happy to answer specific questions or discuss how to improve a protocol. > > Monika > > Monika Beck Gooz, MD, PhD > Research Associate Professor > Department of Drug Discovery & Biomedical Sciences Manager, Cell and > Molecular Imaging Core Hollings Cancer Center > Office: Drug Discovery Building DD507 > Medical University of South Carolina > 70 President Street, MSC 139 > Charleston, SC 29425 > E-mail: [hidden email] > Tel: (843) 876-2363 > > > > -----Original Message----- > From: Confocal Microscopy List > [[hidden email]] On Behalf Of Knecht, David > Sent: Wednesday, October 19, 2016 9:29 AM > To: [hidden email] > Subject: Proximity Ligation Assay > > > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > I am trying to help a student from another lab who is trying to do > proximity ligation assays to look for protein interactions. Anyone > have experience with this technique? It is always hard to tell from > the literature if an approach like that is robust or has a high > failure rate or is extremely tricky to get to work. Thanks- Dave > > Dr. David Knecht > Professor > Department of Molecular and Cell Biology University of Connecticut > 91 N. Eagleville Rd. > Storrs, CT 06269 > 860-486-2200 > > > > > > ---------------------------------------------------------------------- > --- This message was secured via TLS by MUSC. ------------------------------------------------------------------------- This message was secured via TLS by MUSC. |
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0000001ed7f52e4a-dmarc-request |
Re: Proximity Ligation Assay
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*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
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Dear Sylvie, How can you get a good SNR with an on/off response? This is the problem we had. We got either a few (bright) spots per cell or nothing. For me that is a low signal, given that i would expect a lot of events from the number of receptors on our cells. Can you trust such a signal? We spent a lot of money and did not get conclusive answers. Best wishes Andreas From: [hidden email] Sent: 20/10/2016 21:16 To: [hidden email] Subject: Re: Proximity Ligation Assay Same experience here.
We find PLA easier than fret. PLA has a much better SNR and as said. you do not need overexpression. The disadvantage is that you get an on/off answer, not a gradual answer where the molecular distance (between the fluorophores) may be deducted. Additionally, crap in, crap out. With a poor antibody, you get pour PLA. Med vänlig hälsning / Best regards Sylvie @@@@@@@@@@@@@@@@@@@@@@@@ Sylvie Le Guyader, PhD Live Cell Imaging Facility Manager Karolinska Institutet- Bionut Hälsovägen 7, Novum, G lift, floor 6 <a href="tel:14157">14157 Huddinge Sweden mobile: <a href="tel:+46 (0) 73 733 5008">+46 (0) 73 733 5008 office: <a href="tel:+46 (0) 08-524 811 72">+46 (0) 08-524 811 72 LCI website ---- Gooz, Monika wrote ---- *****
To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi David, I did co-localization studies parallel to PLA to prove that they show similar result. I also did FRET and found that PLA is less challenging than FRET. One very important difference between PLA and FRET is that with FRET you have to overexpress proteins that may or may not fold/transport/localize in the cells the way the naturally occurring ones. PLA can show interaction between endogenous proteins and that is a big advantage. Monika Monika Beck Gooz, MD, PhD Research Associate Professor Department of Drug Discovery & Biomedical Sciences Manager, Cell and Molecular Imaging Core Hollings Cancer Center Office: Drug Discovery Building DD507 Medical University of South Carolina 70 President Street, MSC 139 Charleston, SC 29425 E-mail: [hidden email] Tel: (843) 876-2363 Reminder: Please acknowledge your use of the Cell & Molecular Imaging Shared Resource in your publications. Suggested language is as follows: “Supported in part by the Cell & Molecular Imaging Shared Resource, Hollings Cancer Center, Medical University of South Carolina (P30 CA138313) and the Shared Instrumentation Grant S10 OD018113”. Thank you. -----Original Message----- From: Confocal Microscopy List [[hidden email]] On Behalf Of Knecht, David Sent: Thursday, October 20, 2016 10:58 AM To: [hidden email] Subject: Re: Proximity Ligation Assay ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** One of my key questions is people’s experience with how good of a technique this is for detecting protein-protein interaction, which I presume is its general application. Would one use it instead of FRET-FLIM or other co-localization techniques or in addition to? Is it more/less challenging? Does it give the same answers in general? Thanks- Dave Dr. David Knecht Professor Department of Molecular and Cell Biology University of Connecticut 91 N. Eagleville Rd. Storrs, CT 06269 860-486-2200 > On Oct 20, 2016, at 8:37 AM, Cole, Sara <[hidden email]> wrote: > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Hi Dave, > > I've also learned that it is important to image very soon after the procedure because the signal degrades rather quickly with time. Same-day imaging is ideal. > > Sara > > > Sara Cole, PhD > Assistant Director > Senior Microscopist > Campus Microscopy and Imaging Facility (CMIF) > 277 Biomedical Research Tower, 460 West 12th Ave., Columbus, OH 43210 > (614) 292-2814 Office / (614) 688-5564 Lab / (614) 292-4628 Fax > [hidden email] cmif.osu.edu > > > > -----Original Message----- > From: Confocal Microscopy List > [[hidden email]] On Behalf Of Gooz, Monika > Sent: Wednesday, October 19, 2016 2:49 PM > To: [hidden email] > Subject: Re: Proximity Ligation Assay > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Hello David, > > I have a lot of experience with the Duolink PLA. And yes, the signal is robust with good antibodies, can be tricky as well- like every immunostaining procedure. Tissues sometimes give higher background and washing steps are critical. Happy to answer specific questions or discuss how to improve a protocol. > > Monika > > Monika Beck Gooz, MD, PhD > Research Associate Professor > Department of Drug Discovery & Biomedical Sciences Manager, Cell and > Molecular Imaging Core Hollings Cancer Center > Office: Drug Discovery Building DD507 > Medical University of South Carolina > 70 President Street, MSC 139 > Charleston, SC 29425 > E-mail: [hidden email] > Tel: (843) 876-2363 > > > > -----Original Message----- > From: Confocal Microscopy List > [[hidden email]] On Behalf Of Knecht, David > Sent: Wednesday, October 19, 2016 9:29 AM > To: [hidden email] > Subject: Proximity Ligation Assay > > > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > I am trying to help a student from another lab who is trying to do > proximity ligation assays to look for protein interactions. Anyone > have experience with this technique? It is always hard to tell from > the literature if an approach like that is robust or has a high > failure rate or is extremely tricky to get to work. Thanks- Dave > > Dr. David Knecht > Professor > Department of Molecular and Cell Biology University of Connecticut > 91 N. Eagleville Rd. > Storrs, CT 06269 > 860-486-2200 > > > > > > ---------------------------------------------------------------------- > --- This message was secured via TLS by MUSC. ------------------------------------------------------------------------- This message was secured via TLS by MUSC. |
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In reply to this post by Sylvie Le Guyader
*****
To join, leave or search the confocal microscopy listserv, go to:
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Post images on http://www.imgur.com and include the link in your posting.
*****
An open question about PLA is the hit rate - what fraction of complexes are actually appear as a spot, the number per cell seems to be remarkably low.
From: Confocal Microscopy List [[hidden email]] on behalf of Sylvie Le Guyader [[hidden email]]
Sent: 20 October 2016 22:09 To: [hidden email] Subject: Re: Proximity Ligation Assay ***** To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on
http://www.imgur.com and include the link in your posting. *****
Same experience here.
We find PLA easier than fret. PLA has a much better SNR and as said. you do not need overexpression. The disadvantage is that you get an on/off answer, not a gradual answer where the molecular distance (between the fluorophores) may be deducted. Additionally, crap in, crap out. With a poor antibody, you get pour PLA. Med vänlig hälsning / Best regards Sylvie @@@@@@@@@@@@@@@@@@@@@@@@ Sylvie Le Guyader, PhD Live Cell Imaging Facility Manager Karolinska Institutet- Bionut Hälsovägen 7, Novum, G lift, floor 6 <a href="tel:14157" target="_blank">14157 Huddinge Sweden mobile: <a href="tel:+46 (0) 73 733 5008" target="_blank">+46 (0) 73 733 5008 office: <a href="tel:+46 (0) 08-524 811 72" target="_blank">+46 (0) 08-524 811 72 LCI website ---- Gooz, Monika wrote ---- *****
To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi David, I did co-localization studies parallel to PLA to prove that they show similar result. I also did FRET and found that PLA is less challenging than FRET. One very important difference between PLA and FRET is that with FRET you have to overexpress proteins that may or may not fold/transport/localize in the cells the way the naturally occurring ones. PLA can show interaction between endogenous proteins and that is a big advantage. Monika Monika Beck Gooz, MD, PhD Research Associate Professor Department of Drug Discovery & Biomedical Sciences Manager, Cell and Molecular Imaging Core Hollings Cancer Center Office: Drug Discovery Building DD507 Medical University of South Carolina 70 President Street, MSC 139 Charleston, SC 29425 E-mail: [hidden email] Tel: (843) 876-2363 Reminder: Please acknowledge your use of the Cell & Molecular Imaging Shared Resource in your publications. Suggested language is as follows: “Supported in part by the Cell & Molecular Imaging Shared Resource, Hollings Cancer Center, Medical University of South Carolina (P30 CA138313) and the Shared Instrumentation Grant S10 OD018113”. Thank you. -----Original Message----- From: Confocal Microscopy List [[hidden email]] On Behalf Of Knecht, David Sent: Thursday, October 20, 2016 10:58 AM To: [hidden email] Subject: Re: Proximity Ligation Assay ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** One of my key questions is people’s experience with how good of a technique this is for detecting protein-protein interaction, which I presume is its general application. Would one use it instead of FRET-FLIM or other co-localization techniques or in addition to? Is it more/less challenging? Does it give the same answers in general? Thanks- Dave Dr. David Knecht Professor Department of Molecular and Cell Biology University of Connecticut 91 N. Eagleville Rd. Storrs, CT 06269 860-486-2200 > On Oct 20, 2016, at 8:37 AM, Cole, Sara <[hidden email]> wrote: > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Hi Dave, > > I've also learned that it is important to image very soon after the procedure because the signal degrades rather quickly with time. Same-day imaging is ideal. > > Sara > > > Sara Cole, PhD > Assistant Director > Senior Microscopist > Campus Microscopy and Imaging Facility (CMIF) > 277 Biomedical Research Tower, 460 West 12th Ave., Columbus, OH 43210 > (614) 292-2814 Office / (614) 688-5564 Lab / (614) 292-4628 Fax > [hidden email] cmif.osu.edu > > > > -----Original Message----- > From: Confocal Microscopy List > [[hidden email]] On Behalf Of Gooz, Monika > Sent: Wednesday, October 19, 2016 2:49 PM > To: [hidden email] > Subject: Re: Proximity Ligation Assay > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Hello David, > > I have a lot of experience with the Duolink PLA. And yes, the signal is robust with good antibodies, can be tricky as well- like every immunostaining procedure. Tissues sometimes give higher background and washing steps are critical. Happy to answer specific questions or discuss how to improve a protocol. > > Monika > > Monika Beck Gooz, MD, PhD > Research Associate Professor > Department of Drug Discovery & Biomedical Sciences Manager, Cell and > Molecular Imaging Core Hollings Cancer Center > Office: Drug Discovery Building DD507 > Medical University of South Carolina > 70 President Street, MSC 139 > Charleston, SC 29425 > E-mail: [hidden email] > Tel: (843) 876-2363 > > > > -----Original Message----- > From: Confocal Microscopy List > [[hidden email]] On Behalf Of Knecht, David > Sent: Wednesday, October 19, 2016 9:29 AM > To: [hidden email] > Subject: Proximity Ligation Assay > > > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > I am trying to help a student from another lab who is trying to do > proximity ligation assays to look for protein interactions. Anyone > have experience with this technique? It is always hard to tell from > the literature if an approach like that is robust or has a high > failure rate or is extremely tricky to get to work. Thanks- Dave > > Dr. David Knecht > Professor > Department of Molecular and Cell Biology University of Connecticut > 91 N. Eagleville Rd. > Storrs, CT 06269 > 860-486-2200 > > > > > > ---------------------------------------------------------------------- > --- This message was secured via TLS by MUSC. ------------------------------------------------------------------------- This message was secured via TLS by MUSC. |
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Sylvie Le Guyader |
Re: Proximity Ligation Assay
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*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****
Hi again
That's a really good question Jeremy. And formulated slightly differently, it is valid for Fret with overexposed molecules: how much of the fret signal really represents what happens in the cell without massive overexpression of highly modified proteins? Andrea what I mean by SNR is that when the 2 antibodies are not close enough to each other there is no signal at all so you it is easy to image single events because of the high contast. The experience of our users clearly points towards the fact that you need excellent and very specific antibodies though. I have seen lots of antibody pairs give tons of dots per cell. As always things are much easier if you compare treated vs non -treated or Wt vs mutant than if you are going for absolute numbers. One thing that would be interesting to know would be if within a cell population, the reproducibility between cells (e.g. spread of the data cloud) is similar with PLA and Fret. Does anyone know if there is such a study published? Med vänlig hälsning / Best regards Sylvie @@@@@@@@@@@@@@@@@@@@@@@@ Sylvie Le Guyader, PhD Live Cell Imaging Facility Manager Karolinska Institutet- Bionut Hälsovägen 7, Novum, G lift, floor 6 <a href="tel:14157">14157 Huddinge Sweden mobile: <a href="tel:+46 (0) 73 733 5008">+46 (0) 73 733 5008 office: <a href="tel:+46 (0) 08-524 811 72">+46 (0) 08-524 811 72 LCI website ---- Jeremy Adler wrote ---- ***** To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on
http://www.imgur.com and include the link in your posting. *****
An open question about PLA is the hit rate - what fraction of complexes are actually appear as a spot, the number per cell seems to be remarkably low.
From: Confocal Microscopy List [[hidden email]] on behalf of Sylvie Le Guyader [[hidden email]]
Sent: 20 October 2016 22:09 To: [hidden email] Subject: Re: Proximity Ligation Assay ***** To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on
http://www.imgur.com and include the link in your posting. *****
Same experience here.
We find PLA easier than fret. PLA has a much better SNR and as said. you do not need overexpression. The disadvantage is that you get an on/off answer, not a gradual answer where the molecular distance (between the fluorophores) may be deducted. Additionally, crap in, crap out. With a poor antibody, you get pour PLA. Med vänlig hälsning / Best regards Sylvie @@@@@@@@@@@@@@@@@@@@@@@@ Sylvie Le Guyader, PhD Live Cell Imaging Facility Manager Karolinska Institutet- Bionut Hälsovägen 7, Novum, G lift, floor 6 <a href="tel:14157" target="_blank">14157 Huddinge Sweden mobile: <a href="tel:+46 (0) 73 733 5008" target="_blank">+46 (0) 73 733 5008 office: <a href="tel:+46 (0) 08-524 811 72" target="_blank">+46 (0) 08-524 811 72 LCI website ---- Gooz, Monika wrote ---- *****
To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi David, I did co-localization studies parallel to PLA to prove that they show similar result. I also did FRET and found that PLA is less challenging than FRET. One very important difference between PLA and FRET is that with FRET you have to overexpress proteins that may or may not fold/transport/localize in the cells the way the naturally occurring ones. PLA can show interaction between endogenous proteins and that is a big advantage. Monika Monika Beck Gooz, MD, PhD Research Associate Professor Department of Drug Discovery & Biomedical Sciences Manager, Cell and Molecular Imaging Core Hollings Cancer Center Office: Drug Discovery Building DD507 Medical University of South Carolina 70 President Street, MSC 139 Charleston, SC 29425 E-mail: [hidden email] Tel: (843) 876-2363 Reminder: Please acknowledge your use of the Cell & Molecular Imaging Shared Resource in your publications. Suggested language is as follows: “Supported in part by the Cell & Molecular Imaging Shared Resource, Hollings Cancer Center, Medical University of South Carolina (P30 CA138313) and the Shared Instrumentation Grant S10 OD018113”. Thank you. -----Original Message----- From: Confocal Microscopy List [[hidden email]] On Behalf Of Knecht, David Sent: Thursday, October 20, 2016 10:58 AM To: [hidden email] Subject: Re: Proximity Ligation Assay ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** One of my key questions is people’s experience with how good of a technique this is for detecting protein-protein interaction, which I presume is its general application. Would one use it instead of FRET-FLIM or other co-localization techniques or in addition to? Is it more/less challenging? Does it give the same answers in general? Thanks- Dave Dr. David Knecht Professor Department of Molecular and Cell Biology University of Connecticut 91 N. Eagleville Rd. Storrs, CT 06269 860-486-2200 > On Oct 20, 2016, at 8:37 AM, Cole, Sara <[hidden email]> wrote: > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Hi Dave, > > I've also learned that it is important to image very soon after the procedure because the signal degrades rather quickly with time. Same-day imaging is ideal. > > Sara > > > Sara Cole, PhD > Assistant Director > Senior Microscopist > Campus Microscopy and Imaging Facility (CMIF) > 277 Biomedical Research Tower, 460 West 12th Ave., Columbus, OH 43210 > (614) 292-2814 Office / (614) 688-5564 Lab / (614) 292-4628 Fax > [hidden email] cmif.osu.edu > > > > -----Original Message----- > From: Confocal Microscopy List > [[hidden email]] On Behalf Of Gooz, Monika > Sent: Wednesday, October 19, 2016 2:49 PM > To: [hidden email] > Subject: Re: Proximity Ligation Assay > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Hello David, > > I have a lot of experience with the Duolink PLA. And yes, the signal is robust with good antibodies, can be tricky as well- like every immunostaining procedure. Tissues sometimes give higher background and washing steps are critical. Happy to answer specific questions or discuss how to improve a protocol. > > Monika > > Monika Beck Gooz, MD, PhD > Research Associate Professor > Department of Drug Discovery & Biomedical Sciences Manager, Cell and > Molecular Imaging Core Hollings Cancer Center > Office: Drug Discovery Building DD507 > Medical University of South Carolina > 70 President Street, MSC 139 > Charleston, SC 29425 > E-mail: [hidden email] > Tel: (843) 876-2363 > > > > -----Original Message----- > From: Confocal Microscopy List > [[hidden email]] On Behalf Of Knecht, David > Sent: Wednesday, October 19, 2016 9:29 AM > To: [hidden email] > Subject: Proximity Ligation Assay > > > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > I am trying to help a student from another lab who is trying to do > proximity ligation assays to look for protein interactions. Anyone > have experience with this technique? It is always hard to tell from > the literature if an approach like that is robust or has a high > failure rate or is extremely tricky to get to work. Thanks- Dave > > Dr. David Knecht > Professor > Department of Molecular and Cell Biology University of Connecticut > 91 N. Eagleville Rd. > Storrs, CT 06269 > 860-486-2200 > > > > > > ---------------------------------------------------------------------- > --- This message was secured via TLS by MUSC. ------------------------------------------------------------------------- This message was secured via TLS by MUSC. |
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0000001ed7f52e4a-dmarc-request |
Re: Proximity Ligation Assay
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*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****
You can have very good antibodies, but that might not help when the structures you want to label are not accessible, you still will miss most of the complexes. We seem to see the same in STORM. I don't think there can be a specific nanobody for every protein we wish to image. Best wishes Andreas From: [hidden email] Sent: 21/10/2016 07:28 To: [hidden email] Subject: Re: Proximity Ligation Assay That's a really good question Jeremy. And formulated slightly differently, it is valid for Fret with overexposed molecules: how much of the fret signal really represents what happens in the cell without massive overexpression of highly modified proteins? Andrea what I mean by SNR is that when the 2 antibodies are not close enough to each other there is no signal at all so you it is easy to image single events because of the high contast. The experience of our users clearly points towards the fact that you need excellent and very specific antibodies though. I have seen lots of antibody pairs give tons of dots per cell. As always things are much easier if you compare treated vs non -treated or Wt vs mutant than if you are going for absolute numbers. One thing that would be interesting to know would be if within a cell population, the reproducibility between cells (e.g. spread of the data cloud) is similar with PLA and Fret. Does anyone know if there is such a study published? Med vänlig hälsning / Best regards Sylvie @@@@@@@@@@@@@@@@@@@@@@@@ Sylvie Le Guyader, PhD Live Cell Imaging Facility Manager Karolinska Institutet- Bionut Hälsovägen 7, Novum, G lift, floor 6 <a href="tel:14157">14157 Huddinge Sweden mobile: <a href="tel:+46 (0) 73 733 5008">+46 (0) 73 733 5008 office: <a href="tel:+46 (0) 08-524 811 72">+46 (0) 08-524 811 72 LCI website ---- Jeremy Adler wrote ---- ***** To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on
http://www.imgur.com and include the link in your posting. *****
An open question about PLA is the hit rate - what fraction of complexes are actually appear as a spot, the number per cell seems to be remarkably low.
From: Confocal Microscopy List [[hidden email]] on behalf of Sylvie Le Guyader [[hidden email]]
Sent: 20 October 2016 22:09 To: [hidden email] Subject: Re: Proximity Ligation Assay ***** To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on
http://www.imgur.com and include the link in your posting. *****
Same experience here.
We find PLA easier than fret. PLA has a much better SNR and as said. you do not need overexpression. The disadvantage is that you get an on/off answer, not a gradual answer where the molecular distance (between the fluorophores) may be deducted. Additionally, crap in, crap out. With a poor antibody, you get pour PLA. Med vänlig hälsning / Best regards Sylvie @@@@@@@@@@@@@@@@@@@@@@@@ Sylvie Le Guyader, PhD Live Cell Imaging Facility Manager Karolinska Institutet- Bionut Hälsovägen 7, Novum, G lift, floor 6 <a href="tel:14157" target="_blank">14157 Huddinge Sweden mobile: <a href="tel:+46 (0) 73 733 5008" target="_blank">+46 (0) 73 733 5008 office: <a href="tel:+46 (0) 08-524 811 72" target="_blank">+46 (0) 08-524 811 72 LCI website ---- Gooz, Monika wrote ---- *****
To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi David, I did co-localization studies parallel to PLA to prove that they show similar result. I also did FRET and found that PLA is less challenging than FRET. One very important difference between PLA and FRET is that with FRET you have to overexpress proteins that may or may not fold/transport/localize in the cells the way the naturally occurring ones. PLA can show interaction between endogenous proteins and that is a big advantage. Monika Monika Beck Gooz, MD, PhD Research Associate Professor Department of Drug Discovery & Biomedical Sciences Manager, Cell and Molecular Imaging Core Hollings Cancer Center Office: Drug Discovery Building DD507 Medical University of South Carolina 70 President Street, MSC 139 Charleston, SC 29425 E-mail: [hidden email] Tel: (843) 876-2363 Reminder: Please acknowledge your use of the Cell & Molecular Imaging Shared Resource in your publications. Suggested language is as follows: “Supported in part by the Cell & Molecular Imaging Shared Resource, Hollings Cancer Center, Medical University of South Carolina (P30 CA138313) and the Shared Instrumentation Grant S10 OD018113”. Thank you. -----Original Message----- From: Confocal Microscopy List [[hidden email]] On Behalf Of Knecht, David Sent: Thursday, October 20, 2016 10:58 AM To: [hidden email] Subject: Re: Proximity Ligation Assay ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** One of my key questions is people’s experience with how good of a technique this is for detecting protein-protein interaction, which I presume is its general application. Would one use it instead of FRET-FLIM or other co-localization techniques or in addition to? Is it more/less challenging? Does it give the same answers in general? Thanks- Dave Dr. David Knecht Professor Department of Molecular and Cell Biology University of Connecticut 91 N. Eagleville Rd. Storrs, CT 06269 860-486-2200 > On Oct 20, 2016, at 8:37 AM, Cole, Sara <[hidden email]> wrote: > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Hi Dave, > > I've also learned that it is important to image very soon after the procedure because the signal degrades rather quickly with time. Same-day imaging is ideal. > > Sara > > > Sara Cole, PhD > Assistant Director > Senior Microscopist > Campus Microscopy and Imaging Facility (CMIF) > 277 Biomedical Research Tower, 460 West 12th Ave., Columbus, OH 43210 > (614) 292-2814 Office / (614) 688-5564 Lab / (614) 292-4628 Fax > [hidden email] cmif.osu.edu > > > > -----Original Message----- > From: Confocal Microscopy List > [[hidden email]] On Behalf Of Gooz, Monika > Sent: Wednesday, October 19, 2016 2:49 PM > To: [hidden email] > Subject: Re: Proximity Ligation Assay > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Hello David, > > I have a lot of experience with the Duolink PLA. And yes, the signal is robust with good antibodies, can be tricky as well- like every immunostaining procedure. Tissues sometimes give higher background and washing steps are critical. Happy to answer specific questions or discuss how to improve a protocol. > > Monika > > Monika Beck Gooz, MD, PhD > Research Associate Professor > Department of Drug Discovery & Biomedical Sciences Manager, Cell and > Molecular Imaging Core Hollings Cancer Center > Office: Drug Discovery Building DD507 > Medical University of South Carolina > 70 President Street, MSC 139 > Charleston, SC 29425 > E-mail: [hidden email] > Tel: (843) 876-2363 > > > > -----Original Message----- > From: Confocal Microscopy List > [[hidden email]] On Behalf Of Knecht, David > Sent: Wednesday, October 19, 2016 9:29 AM > To: [hidden email] > Subject: Proximity Ligation Assay > > > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > I am trying to help a student from another lab who is trying to do > proximity ligation assays to look for protein interactions. Anyone > have experience with this technique? It is always hard to tell from > the literature if an approach like that is robust or has a high > failure rate or is extremely tricky to get to work. Thanks- Dave > > Dr. David Knecht > Professor > Department of Molecular and Cell Biology University of Connecticut > 91 N. Eagleville Rd. > Storrs, CT 06269 > 860-486-2200 > > > > > > ---------------------------------------------------------------------- > --- This message was secured via TLS by MUSC. ------------------------------------------------------------------------- This message was secured via TLS by MUSC. |
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George McNamara |
Re: Proximity Ligation Assay ... you might also consider BirA or APEX2 peroxidase tyramide
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In reply to this post by Knecht, David
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi David, you might also consider BirA or APEX2 peroxidase tyramide recent BirA paper (chromatin, would be generalizable) is (first URl is to a blog entry about the article -- article is open access): https://www.linkedin.com/pulse/determination-local-chromatin-composition-casid-nechyporuk-zloy http://www.tandfonline.com/doi/full/10.1080/19491034.2016.1239000 Chromatin structure and function are determined by a plethora of proteins whose genome-wide distribution is typically assessed by immunoprecipitation (ChIP). Here, we developed a novel tool to investigate the local chromatin environment at specific DNA sequences. We combined the programmable DNA binding of dCas9 with the promiscuous biotin ligase BirA* (CasID) to biotinylate proteins in the direct vicinity of specific loci. Subsequent streptavidin-mediated precipitation and mass spectrometry identified both known and previously unknown chromatin factors associated with repetitive telomeric, major satellite and minor satellite DNA. With super-resolution microscopy, we confirmed the localization of the putative transcription factor ZNF512 at chromocenters. The versatility of CasID facilitates the systematic elucidation of functional protein complexes and locus-specific chromatin composition. APEX2 papers: Spatially resolved proteomic mapping in living cells with the engineered peroxidase APEX2. Hung V, Udeshi ND, Lam SS, Loh KH, Cox KJ, Pedram K, Carr SA, Ting AY. Nat Protoc. 2016 Mar;11(3):456-75. doi: 10.1038/nprot.2016.018. Epub 2016 Feb 11. PMID: 26866790 Free PMC Article Directed evolution of APEX2 for electron microscopy and proximity labeling. Lam SS, Martell JD, Kamer KJ, Deerinck TJ, Ellisman MH, Mootha VK, Ting AY. Nat Methods. 2015 Jan;12(1):51-4. doi: 10.1038/nmeth.3179. Epub 2014 Nov 24. PMID: 25419960 // as for FRET approaches using antibodies with fluorescent dyes, Janos Szollosi and colleagues have a long time track record reporting that Fabs (~1 nm long) should be used, not IgG (~10 nm Y-shaped) (and don't forget the controls!): https://www.ncbi.nlm.nih.gov/pubmed/?term=szollosi+j+fret+fab Clustering of class I HLA oligomers with CD8 and TCR: three-dimensional models based on fluorescence resonance energy transfer and crystallographic data. Gáspár R Jr, Bagossi P, Bene L, Matkó J, Szöllosi J, Tozsér J, Fésüs L, Waldmann TA, Damjanovich S. J Immunol. 2001 Apr 15;166(8):5078-86. PMID: 11290789 EGF-induced redistribution of erbB2 on breast tumor cells: flow and image cytometric energy transfer measurements. Nagy P, Bene L, Balázs M, Hyun WC, Lockett SJ, Chiang NY, Waldman F, Feuerstein BG, Damjanovich S, Szöllosi J. Cytometry. 1998 Jun 1;32(2):120-31. PMID: 9627225 Analysis of cell surface molecular distributions and cellular signaling by flow cytometry. Matkó J, Mátyus L, Szöllösi J, Bene L, Jenei A, Nagy P, Bodnár A, Damjanovich S. J Fluoresc. 1994 Dec;4(4):303-14. doi: 10.1007/BF01881445. PMID: 24233604 enjoy, George On 10/20/2016 9:58 AM, Knecht, David wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > One of my key questions is people’s experience with how good of a technique this is for detecting protein-protein interaction, which I presume is its general application. Would one use it instead of FRET-FLIM or other co-localization techniques or in addition to? Is it more/less challenging? Does it give the same answers in general? Thanks- Dave > > Dr. David Knecht > Professor > Department of Molecular and Cell Biology > University of Connecticut > 91 N. Eagleville Rd. > Storrs, CT 06269 > 860-486-2200 > >> On Oct 20, 2016, at 8:37 AM, Cole, Sara <[hidden email]> wrote: >> >> ***** >> To join, leave or search the confocal microscopy listserv, go to: >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >> Post images on http://www.imgur.com and include the link in your posting. >> ***** >> >> Hi Dave, >> >> I've also learned that it is important to image very soon after the procedure because the signal degrades rather quickly with time. Same-day imaging is ideal. >> >> Sara >> >> >> Sara Cole, PhD >> Assistant Director >> Senior Microscopist >> Campus Microscopy and Imaging Facility (CMIF) >> 277 Biomedical Research Tower, 460 West 12th Ave., Columbus, OH 43210 >> (614) 292-2814 Office / (614) 688-5564 Lab / (614) 292-4628 Fax >> [hidden email] cmif.osu.edu >> >> >> >> -----Original Message----- >> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Gooz, Monika >> Sent: Wednesday, October 19, 2016 2:49 PM >> To: [hidden email] >> Subject: Re: Proximity Ligation Assay >> >> ***** >> To join, leave or search the confocal microscopy listserv, go to: >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >> Post images on http://www.imgur.com and include the link in your posting. >> ***** >> >> Hello David, >> >> I have a lot of experience with the Duolink PLA. And yes, the signal is robust with good antibodies, can be tricky as well- like every immunostaining procedure. Tissues sometimes give higher background and washing steps are critical. Happy to answer specific questions or discuss how to improve a protocol. >> >> Monika >> >> Monika Beck Gooz, MD, PhD >> Research Associate Professor >> Department of Drug Discovery & Biomedical Sciences Manager, Cell and Molecular Imaging Core Hollings Cancer Center >> Office: Drug Discovery Building DD507 >> Medical University of South Carolina >> 70 President Street, MSC 139 >> Charleston, SC 29425 >> E-mail: [hidden email] >> Tel: (843) 876-2363 >> >> >> >> -----Original Message----- >> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Knecht, David >> Sent: Wednesday, October 19, 2016 9:29 AM >> To: [hidden email] >> Subject: Proximity Ligation Assay >> >> >> >> ***** >> To join, leave or search the confocal microscopy listserv, go to: >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >> Post images on http://www.imgur.com and include the link in your posting. >> ***** >> >> I am trying to help a student from another lab who is trying to do proximity ligation assays to look for protein interactions. Anyone have experience with this technique? It is always hard to tell from the literature if an approach like that is robust or has a high failure rate or is extremely tricky to get to work. Thanks- Dave >> >> Dr. David Knecht >> Professor >> Department of Molecular and Cell Biology University of Connecticut >> 91 N. Eagleville Rd. >> Storrs, CT 06269 >> 860-486-2200 >> >> >> >> >> >> ------------------------------------------------------------------------- >> This message was secured via TLS by MUSC. -- George McNamara, PhD Houston, TX 77054 [hidden email] https://www.linkedin.com/in/georgemcnamara https://works.bepress.com/gmcnamara/75/ http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/44962650 |
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Vincent Schoonderwoert |
ICS open source file format to be maintained by SVI
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To join, leave or search the confocal microscopy listserv, go to:
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Post images on http://www.imgur.com and include the link in your posting.
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Dear all, We are happy to announce that we at Scientific Volume Imaging (SVI) will be taking over the maintenance of 'libics', the reference library for ICS (Image Cytometry Standard), an open standard for reading and writing microscopy images. The libics library is distributed under the GNU Lesser General Public License (LGPL, version 2.1), which allows it to be used freely in both commercial and open source software. At SVI, we will actively maintain and further develop the libics software to ensure it remains a excellent choice for an open data format to store multi-dimensional data together with their microscopy parameters. We remain committed to making the software available as open source under its current LGPL license, and welcome all feedback, bug reports and contributions. These can be directed to us at: [hidden email]. The source code can be found at GitHub: https://github.com/svi-opensource/libics Online documentation is available at: https://svi-opensource.github.io/libics/ Finally, we would like to express our appreciation for Cris Luengo for work on libics during the past 16 years , and the high quality of the software. You can visit his blog on image analysis here: http://www.crisluengo.net/ Best regards, Vincent ************************************************************** Vincent Schoonderwoert, PhD Senior Imaging Specialist/Account Manager Scientific Volume Imaging www.svi.nl +31 35 642 1626 ************************************************************** Join the Huygens Image Contest 2016. See svi.nl/HuygensImageContest2016 |
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