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Genetic Barcoding with Fluorescent Proteins for Multiplexed Applications
Published on: April 14, 2015
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Solid state fluorescence of proteins in high throughput mode and its applications
Saurabh Gautam1, Munishwar N Gupta1
1Department of Chemistry, Indian Institute of Technology Delhi, New Delhi, 110016, India.
F1000Research
|April 24, 2019
Summary
A simple method allows recording protein fluorescence spectra in solid states, crucial for biocatalysis and sensor applications. Lyophilization caused blue shifts and intensity increases, indicating microenvironment changes.
Area of Science:
- Biochemistry
- Spectroscopy
- Protein Analysis
Background:
- Comparing solution and solid-state protein fluorescence spectra is key for understanding structural changes during drying or immobilization.
- Solid-state analysis is vital for biocatalysis and sensor development.
- Existing methods for solid-state fluorescence often require specialized accessories.
Purpose of the Study:
- To present a simple, high-throughput method for recording protein fluorescence emission spectra from powders without dedicated accessories.
- To validate this method against commercial accessories.
- To investigate protein microenvironment changes in solid states.
Main Methods:
- Utilizing black-coated 96-well plates with top-based excitation and emission paths to minimize scatter and noise.
- Recording fluorescence emission spectra of proteins in solution, precipitated, and lyophilized forms.
- Analyzing spectral shifts (λmax) and intensity changes as indicators of structural and microenvironmental modifications.
Main Results:
- The developed method yielded noise-free spectra comparable to commercial accessories.
- Lyophilization resulted in blue-shifted emission spectra (4-9 nm) and increased fluorescence intensity for studied proteins.
- Analysis of alpha-chymotrypsin and green fluorescent protein (GFP) confirmed microenvironment changes upon lyophilization, including alterations around GFP's cyclic chromophore.
Conclusions:
- The described method offers a straightforward and effective approach for solid-state protein fluorescence spectroscopy.
- Spectral shifts (λmax) are reliable indicators of protein structural changes in solid forms.
- Lyophilization significantly alters the protein microenvironment, impacting fluorescence properties.
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