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.
- Black opaque, clear bottom, flat-bottom 96-well plate (e.g. Corning COSTAR 3603)
- Fresh or thawed protein samples on ice
- Biotek Synergy H1 Plate Reader (or similar)
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.
Protocol > Action > Read Settings for the BioTek Synergy H1 Plate Reader
|Fluorophore||Step label||Detect. Method||Read Type||Read Speed||Wavelengths||Excitation||Emission||Optics Position||Gain|
|RFP or mCherry||RFP||Fluorescence||End Point||Normal||1||580||610||Bottom||100|
Part 2: Measure background protein levels and normalize the fluorescence (IMPORTANT)
- Follow the Bradford Assay procedure.
Helpful tip - to conserve precious samples, you can use 5 μL from each well in Part 1 for the bradford assay, instead of using 5 μL from the original sample tubes.
- 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.
- In your figure (poster, paper, thesis, etc.) report the Fluorescence per μg protein.