This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Immunolocalization of macromolecules is a valuable part of any study that characterizes the roles of specific proteins in cellular processes. However, sample preparation techniques that are designed to yield high quality structural preservation are often not compatible with good antibody labeling. Moreover, immunolabeling protocols that do feature high quality preservation (i.e. low-temperature Lowicryl embedding) may not adequately reveal structures of interest or be useful for a broad range of immunological reagents. On the other hand, techniques that usually yield fine immunolabeling often result in a reduced quality of structural preservation. To study the 3D structure of the Golgi and associated compartments in lactating rat mammary epithelial cells, we have employed the best approaches for each of these issues and then combine their results to address our scientific questions. Our results demonstrate that immunolabeling with a modification of the Tokuyasu method can be correlated with thin-section and tomographic images from well-preserved specimens prepared by high-pressure freezing and freeze-substitution. The result is an improvement in the accuracy of the localization of proteins and greater understanding of the organization of membrane structures involved in milk secretion.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
2P41RR000592-39
Application #
7955029
Study Section
Special Emphasis Panel (ZRG1-CB-J (40))
Project Start
2009-08-01
Project End
2010-04-30
Budget Start
2009-08-01
Budget End
2010-04-30
Support Year
39
Fiscal Year
2009
Total Cost
$10,719
Indirect Cost
Name
University of Colorado at Boulder
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
007431505
City
Boulder
State
CO
Country
United States
Zip Code
80309
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