Posted by ocho0020 on May 8, 2008 07:40 AM|Permalink
Comments
One thing I'm a bit confused about is about how the mutant fluorescent proteins are made. Is just the chromaphore modified or is the entire helix altered? The paper says that a mutagenesis of the EGFP is done, but what exactly is this changing?
1. Can you comment on the z-resolution? Does the limiting resolution match the separation between z-stacks?
2. You mentioned that two different spectral properties of the mutants make it possible to identify interactions. How does one know that two molecules are truly interacting and not just nearby? Does the microscope used have sufficient resolution to observe two molecules side by side?
3. What filters would you select to determine FRET?
4. Please elaborate on the cross-over measurements.
Melissa:
The authors said they received the W7 mutant from a colleague and made the 10C mutant through site-directed mutagenesis. In site-directed mutagenesis a single (or multiple) amino acid is changed to a different amino acid. This is accomplished by mutating a single base pair in the DNA sequence that encodes the protein; often this is done using PCR and primers that have the modified base pair. So if they say, for instance, S65G it means they mutated a serine at position 65 to glycine.
As far as the chromophore goes, it consists of residues 65, 66, and 67. The 10C and W7 mutants only change one of these amino acids, the other changes are just spatially close to they chromophore and change the fluorescence emission through other electronic interactions.
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Comments
One thing I'm a bit confused about is about how the mutant fluorescent proteins are made. Is just the chromaphore modified or is the entire helix altered? The paper says that a mutagenesis of the EGFP is done, but what exactly is this changing?
Posted by: Melissa Maurer-Jones | May 8, 2008 12:15 PM
1. Can you comment on the z-resolution? Does the limiting resolution match the separation between z-stacks?
2. You mentioned that two different spectral properties of the mutants make it possible to identify interactions. How does one know that two molecules are truly interacting and not just nearby? Does the microscope used have sufficient resolution to observe two molecules side by side?
3. What filters would you select to determine FRET?
4. Please elaborate on the cross-over measurements.
4.
Posted by: Edgar Arriaga | May 8, 2008 04:08 PM
Melissa:
The authors said they received the W7 mutant from a colleague and made the 10C mutant through site-directed mutagenesis. In site-directed mutagenesis a single (or multiple) amino acid is changed to a different amino acid. This is accomplished by mutating a single base pair in the DNA sequence that encodes the protein; often this is done using PCR and primers that have the modified base pair. So if they say, for instance, S65G it means they mutated a serine at position 65 to glycine.
As far as the chromophore goes, it consists of residues 65, 66, and 67. The 10C and W7 mutants only change one of these amino acids, the other changes are just spatially close to they chromophore and change the fluorescence emission through other electronic interactions.
Posted by: Josh Ochocki | May 8, 2008 05:54 PM