Belmont Lab
 Unversity of Illinois at Urbana-Champaign
Alumni Labs
 
In vivo immunogold labeling

A problem in general for immunogold labeling is how to preserve good ultrastructure while obtaining efficient, 3-dimensional labeling. Pre-embedding immunogold labeling often requires detergent permeabilization to allow the gold labeled antibodies to penetrate cell structures. In our experiments we noticed that using fixation conditions which roughly preserved in vivo levels of chromatin compaction resulted in low efficiency immunogold labeling restricted to the surfaces of the condensed chromatin. This greatly restricted the sensitivity of labeling of lac operator tagged chromatin, under conditions which already noticeably perturbed chromatin and nuclear ultrastructure. In contrast post-embedding immunogold labeling typically results in very low efficiency labeling restricted to the surface of the plastic sections.

We have recently demonstrated that microinjection of Nangold labeled IgG or Fab' fragments results in high efficiency labeling of chromatin which penetrates condensed large-scale chromatin structures. Because cells can be fixed directly with strong fixatives, ultrastructural preservation is significantly improved. Initially our work used anti-lac repressor antibodies but using antibodies against GFP we were able to extend this methodology to the localization of a number of nuclear proteins.

Selected Publications:

Hu Y, Kireev I, Plutz M, Ashourian N, Belmont AS. Large-scale chromatin structure of inducible genes: transcription on a condensed, linear template. J Cell Biol. 2009 Apr 6; 185 (1) :87-100. PubMed PMID:19349581; PubMed Central PMCID: PMC2700507.

Kireev I, Lakonishok M, Liu W, Joshi VN, Powell R, Belmont AS. In vivo immunogold labeling confirms large-scale chromatin folding motifs. Nat Methods. 2008 Apr; 5 (4) :311-3. PubMed PMID:18345005.

 

 

 

 

 

 

 

 

 

 

 

       
 
Copyright @ 2009 Belmont Lab. All rights reserved.
 
 
Belmont Lab  | B107 CLSL,601 S. Goodwin Ave, Urbana, IL 61801 |