Haynes:ExtFluorProtein: Difference between revisions
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= | =Fluorescent Protein Measurement= | ||
by Karmella Haynes, 2014<br><br> | by Karmella Haynes, 2014<br><br> | ||
Principle: Fluorescent proteins that are gently extracted from cell samples can be detected on a plat reader. This procedure is for determining the yield of fluorescent protein relative to background protein levels. | Principle: Fluorescent proteins that are gently extracted from cell samples can be detected on a plat reader. This procedure is for determining the yield of fluorescent protein relative to background protein levels. | ||
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PROCEDURE | PROCEDURE | ||
Part 1: Fluorescent protein measurement | '''Part 1: Fluorescent protein measurement''' | ||
# Follow the protein extraction protocol [link]. The final volume will be ~500 μL for 5x10<sup>6</sup> cells or less. The samples should include a negative control: non-fluorescent cells. | # Follow the protein extraction protocol [link]. The final volume will be ~500 μL for 5x10<sup>6</sup> cells or less. The samples should include a negative control: non-fluorescent cells. | ||
# Transfer 100 μL of each sample onto a well int eh 96-well plate. Avoid making bubbles. | # Transfer 100 μL of each sample onto a well int eh 96-well plate. Avoid making bubbles. | ||
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Part 2: Measure background protein levels and normalize the | '''Settings for the BioTek Synergy H1 Plate Reader''' | ||
{| | |||
|- | |||
| Fluorophore || Mode || Excitation || Emission || Read from... | |||
|- | |||
| RFP/ mCherry || Fluoresence || ### || ### || Bottom | |||
'''Part 2: Measure background protein levels and normalize the fluorescence (IMPORTANT)''' | |||
# Follow the [http://openwetware.org/wiki/Haynes:Bradford Bradford Assay] procedure. | # Follow the [http://openwetware.org/wiki/Haynes:Bradford Bradford Assay] procedure. | ||
# Use the calculated protein level from the Bradford procedure to normalize the fluorescence values: (fluorescence signal in 100 uL) / (calculated protein μg/μL * 100 μL) = Fluorescence per μg total protein. | # Use the calculated protein level from the Bradford procedure to normalize the fluorescence values: (fluorescence signal in 100 uL) / (calculated protein μg/μL * 100 μL) = Fluorescence per μg total protein. | ||
Revision as of 12:26, 15 September 2014
Fluorescent Protein Measurement
by Karmella Haynes, 2014
Principle: Fluorescent proteins that are gently extracted from cell samples can be detected on a plat reader. This procedure is for determining the yield of fluorescent protein relative to background protein levels.
MATERIALS
- Black opaque, clear bottom, flat-bottom 96-well plate (e.g. Corning COSTAR 3603)
EQUIPMENT
- Biotek Synergy H1 Plate Reader (or similar)
PROCEDURE
Part 1: Fluorescent protein measurement
- Follow the protein extraction protocol [link]. The final volume will be ~500 μL for 5x106 cells or less. The samples should include a negative control: non-fluorescent cells.
- Transfer 100 μL of each sample onto a well int eh 96-well plate. Avoid making bubbles.
- Use the table below to set up a New Protocol on the plate reader. If you do not know how to use the plate reader, ask someone to help you to set up the machine.
Settings for the BioTek Synergy H1 Plate Reader
| Fluorophore | Mode | Excitation | Emission | Read from... |
| RFP/ mCherry | Fluoresence | ### | ### | Bottom
Part 2: Measure background protein levels and normalize the fluorescence (IMPORTANT)
|
