Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > Molecular Research Topics Forum > Protocols and Methods Forum
Register Search Today's Posts Mark Forums Read

Protocols and Methods Forum Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


Antibody binding to nonpermeabilized cells

Antibody binding to nonpermeabilized cells - Protocols and Methods Forum

Antibody binding to nonpermeabilized cells - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 11-06-2007, 07:06 PM
Beug, Shawn
Guest
 
Posts: n/a
Default Antibody binding to nonpermeabilized cells



Hi all,



I am interested in calculating the ratio of intracellular and
extracellular GFP-tagged protein in primary neurons. I am trying to
demonstrate that perturbation of posttranslational modification affects
extracellular/intracellular sublocalization. I was wondering if
immunnostaining for transfected neurons with an anti-GFP antibody (using
a Cy3-conjugated secondary antibody) would be the best way to calculate
this ratio. I have done this before in the past but am unsure if
antibodies would stain intracellular proteins, and as a result, give me
the wrong numbers.



Cheers, Shawn




--------------------
Confidentiality Statement - The contents of this e-mail, including its attachment, are intended for the exclusive use of the recipient and may contain confidential or privileged information. If you are not the intended recipient, you are strictly prohibited from reading, using, disclosing, copying, or distributing this e-mail or any of its contents. If you received this e-mail in error, please notify the sender by reply e-mail immediately or the Privacy Office ([Only registered users see links. ].ca ) and permanently delete this e-mail and its attachments, along with any copies thereof. Thank you.

Avis de confidentialité – Ce courriel, y compris ses pièces jointes, s’adresse au destinataire uniquement et pourrait contenir des renseignements confidentiels. Si vous n’êtes pas le bon destinataire, il est strictement interdit de lire, d’utiliser, de divulguer, de copier ou de diffuser ce courriel ou son contenu, en partie ou en entier. Si vous avez reçu ce courriel par erreur, veuillez en informer immédiatement l’expéditeur ou le bureau de la Protection des renseignements personnels ([Only registered users see links. ].ca), puis effacez le courriel ainsi que les pièces jointes et toute autre copie. Merci.
--------------------
Reply With Quote
  #2  
Old 11-15-2007, 01:40 PM
Dr Engelbert Buxbaum
Guest
 
Posts: n/a
Default Antibody binding to nonpermeabilized cells

Am 06.11.2007, 15:06 Uhr, schrieb Beug, Shawn <[Only registered users see links. ]>:


Could be done. The easier way is to work with a non-membranepermeable
fluorescence quencher (iodide, acrylamide or the like). The fluorescence
of extracellular GFP would be quenched, that of intracellular GFP not, the
ratio of fluorescence with/without quencher is, under appropriate
conditions, equal to the ratio of intracellular / total GFP.

See:

@book{Lak-99,
AUTHOR= {J.R. Lakowicz},
TITLE= {Principles of fluorescence spectroscopy},
YEAR= {1999},
PUBLISHER= {Kluwer},
ADDRESS= {New York},
EDITION= {Second},
LANGUAGE= {engl}
}

p.s.: It makes little sense to add a several language confidentiality
statement to a message published on usenet.
Reply With Quote
Reply

Tags
antibody , binding , cells , nonpermeabilized


Thread Tools
Display Modes

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off
Trackbacks are On
Pingbacks are On
Refbacks are On

Forum Jump

Similar Threads
Thread Thread Starter Forum Replies Last Post
mouse hepatocytes isolation amicia Cell Biology and Cell Culture 39 05-11-2012 11:52 AM
Characteristics of cancerous cells aftabac Cell Biology and Cell Culture 2 08-26-2011 11:40 PM
Human Cytome Project - Update 24 Jan. 2005 Peter Van Osta Cell Biology and Cell Culture 1 08-01-2010 02:18 PM
Production of Monoclonal Antibodies - Technique in Mouse Cellbiogal Antibody Forum 2 01-08-2009 10:52 PM
Human Cytome Project - Update 6 Jan. 2005 Peter Van Osta Cell Biology and Cell Culture 0 01-06-2005 11:18 AM


All times are GMT. The time now is 12:20 AM.


Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.15633 seconds with 16 queries