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| Content Provider | World Health Organization (WHO)-Global Index Medicus |
|---|---|
| Author | Wells, Amber L. Condeelis, John S. Singer, Robert H. Zenklusen, Daniel |
| Description | Country affiliation: United States Author Affiliation: Wells AL ( Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.) |
| Abstract | INTRODUCTIONThis protocol describes a method for establishing a green fluorescent protein (GFP) calibration curve using dilutions of recombinant GFP and blue fluorescent beads. The total fluorescence intensity (TFI) per mRNA molecule is first calculated by imaging serial dilutions of purified enhanced GFP (eGFP) to determine the TFI within a specific volume. A calibration curve of fluorescence intensity in a given voxel per molecule of GFP is then used to determine the number of GFP molecules in the sample of formaldehyde fixed cells to be imaged. This is followed by a method for detection of single molecules in formaldehyde-fixed and live cells. These cells have been cotransfected with mRNA reporter and MCP-xFP plasmids, where MCP-xFP refers to a fluorescent protein fused to the MS2 capsid protein. It is important to collect micrographs and establish the calibration curve on the same day that the cells are imaged, using the same equipment configuration, camera settings, and image acquisition parameters. |
| File Format | HTM / HTML |
| ISSN | 19403402 |
| e-ISSN | 15596095 |
| DOI | 10.1101/pdb.prot4871 |
| Journal | Cold Spring Harbor Protocols |
| Volume Number | 2007 |
| Language | English |
| Publisher | Cold Spring Harbor Laboratory Press |
| Publisher Date | 2007-11-01 |
| Publisher Place | United States |
| Access Restriction | Open |
| Subject Keyword | Discipline Clinical Laboratory Techniques |
| Content Type | Text |
| Resource Type | Article |
| Subject | Biochemistry, Genetics and Molecular Biology |
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