questions about FRET

[Ramana Sidhaye]

Hi-

  I am interested in looking at colocalization and/or FRET between two proteins. Unfortunately, the commercially available antibodies for both proteins are not very good, and the antibodies that have been generated by our group and others that do work well are both in rabbit. I was wondering if there was a good protocol or generally accepted mechanism for dealing with this.

 

Thanks,

Ramana Sidhaye

 

 

Venkataramana Sidhaye, MD

Assistant Professor, Pulmonary and Critical Care Medicine

Johns Hopkins University

 

 

[Arne K Christensen]

If the antibodies available to you are purified and in sufficient
quantity and concentration (1 - 2 mg/mL), you can directly label the
antibodies covalently with an appropriate fluorophore.  Fluorophore
conjugate, or labeling kits are available through Invitrogen:

*http://tinyurl.com/mwabcj

* Invitrogen also has strategies for labeling small quantities of
antibody non-covalently.  I haven't have very much luck with one such
kit, called Zenon, but the covalent labeling method has worked well for
me.  Directly labeled antibodies will certainly work for colacalization
studies; however, I'm not sure if  they would be appropriate for FRET.

Arne


Ramana Sidhaye wrote:

>
> *Hi-*
>
> *  I am interested in looking at colocalization and/or FRET between
> two proteins. Unfortunately, the commercially available antibodies for
> both proteins are not very good, and the antibodies that have been
> generated by our group and others that do work well are both in
> rabbit. I was wondering if there was a good protocol or generally
> accepted mechanism for dealing with this.*
>
> * *
>
> *Thanks,*
>
> *Ramana Sidhaye*
>
> * *
>
>  
>
> Venkataramana Sidhaye, MD
>
> Assistant Professor, Pulmonary and Critical Care Medicine
>
> Johns Hopkins University
>
>  
>
>  
>

--

__

Arne K Christensen

Postdoctoral Research Associate
University of Massachusetts, Amherst

USGS Conte Anadromous Fish Research Center
One Migratory Way, PO Box 796
Turners Falls, MA  01376

Email: [hidden email]
Phone: [phone]
  Fax: [phone]
  URL: www.biobog.com

[JOEL B. SHEFFIELD]

You could look into direct labeling of the antibodies that do work.  
Be aware, though, that the size of antibody molecules may create
steric distortions of the actual associations that might be present.

Joel


-------------- Original message ---------------
Hi-
 I am interested in looking at colocalization and/or FRET between
two proteins. Unfortunately, the commercially available antibodies
for both proteins are not very good, and the antibodies that have
been generated by our group and others that do work well are both in
rabbit. I was wondering if there was a good protocol or generally
accepted mechanism for dealing with this.

Thanks,
Ramana Sidhaye


Venkataramana Sidhaye, MD
Assistant Professor, Pulmonary and Critical Care Medicine
Johns Hopkins University



--
Joel B. Sheffield, Ph.D.
Biology Department, Temple University
1900 North 12th Street
Philadelphia, PA 19122
[hidden email]  
[phone], fax [phone]
http://astro.temple.edu/~jbs

[Michael Crow Ph.D.]

I am not familiar with doing FRET using antibodies.  We have used YFP and CFP-tagged proteins to do this.  

 

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Ramana Sidhaye
Sent: Thursday, June 18, 2009 10:50 AM
To: [hidden email]
Subject: questions about FRET

 

Hi-

  I am interested in looking at colocalization and/or FRET between two proteins. Unfortunately, the commercially available antibodies for both proteins are not very good, and the antibodies that have been generated by our group and others that do work well are both in rabbit. I was wondering if there was a good protocol or generally accepted mechanism for dealing with this.

 

Thanks,

Ramana Sidhaye

 

 

Venkataramana Sidhaye, MD

Assistant Professor, Pulmonary and Critical Care Medicine

Johns Hopkins University

 

 

[Periasamy, Ammasi (ap3t)]

Please visit our web site there are few papers on antibody based FRET imaging

http://www.kcci.virginia.edu/Publications/index.php

 

 

Ammasi Periasamy, Ph.D.

Director, Keck Center for Cellular Imaging (KCCI)

Professor of Biology and Biomedical Engineering

Biology, Gilmer Hall (064), McCormick Rd

University of Virginia

Charlottesville, VA 22904

Voice: 434-243-7602 (Office); 982-4869 (lab)

Fax:[phone]; Email:[hidden email]

http//:www.kcci.virginia.edu

************************

Workshop on FRET Microscopy, March 9-13, 2010

http://www.kcci.virginia.edu/workshop/workshop2010/index.php

*************************

 

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Michael Crow Ph.D.
Sent: Thursday, June 18, 2009 1:39 PM
To: [hidden email]
Subject: Re: questions about FRET

 

I am not familiar with doing FRET using antibodies.  We have used YFP and CFP-tagged proteins to do this.  

 

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Ramana Sidhaye
Sent: Thursday, June 18, 2009 10:50 AM
To: [hidden email]
Subject: questions about FRET

 

Hi-

  I am interested in looking at colocalization and/or FRET between two proteins. Unfortunately, the commercially available antibodies for both proteins are not very good, and the antibodies that have been generated by our group and others that do work well are both in rabbit. I was wondering if there was a good protocol or generally accepted mechanism for dealing with this.

 

Thanks,

Ramana Sidhaye

 

 

Venkataramana Sidhaye, MD

Assistant Professor, Pulmonary and Critical Care Medicine

Johns Hopkins University

 

 

[vb-2]

Hi Ramana,

 

Doing FRET using fluorescently tagged antibodies is rather tricky (I would rather use fluorescently tagged monoclonals that reveal high specificity and/or affinity), thus I agree with Michael to rather utilize Cerulean FP and EYFP, both with monomeric mutations (A206K at least, that is already within the CeFP sequence but not in the EYFP; all three monomeric mutations might be a plus - A206K, L221K, F223R, as hetero-aggregation of GFP-derived proteins is very common than rare; … but many Assistant to Associate Professors are under strong pressure to deny this unfortunate “stickiness” of FPs till they “grow up” to the Full Professor title).

 

Good luck,

 

Vitaly

NCI-Frederick,

301-846-6575

 

 

 

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Michael Crow Ph.D.
Sent: Thursday, June 18, 2009 12:39 PM
To: [hidden email]
Subject: Re: questions about FRET

 

I am not familiar with doing FRET using antibodies.  We have used YFP and CFP-tagged proteins to do this.  

 

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Ramana Sidhaye
Sent: Thursday, June 18, 2009 10:50 AM
To: [hidden email]
Subject: questions about FRET

 

Hi-

  I am interested in looking at colocalization and/or FRET between two proteins. Unfortunately, the commercially available antibodies for both proteins are not very good, and the antibodies that have been generated by our group and others that do work well are both in rabbit. I was wondering if there was a good protocol or generally accepted mechanism for dealing with this.

 

Thanks,

Ramana Sidhaye

 

 

Venkataramana Sidhaye, MD

Assistant Professor, Pulmonary and Critical Care Medicine

Johns Hopkins University