Haynes:ExtFluorProtein

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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)
  • Fresh or thawed protein samples on ice


EQUIPMENT

  • Biotek Synergy H1 Plate Reader (or similar)


PROCEDURE

Part 1: Fluorescent protein measurement

  1. 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.
  2. Transfer 100 μL of each sample onto a well int eh 96-well plate. Avoid making bubbles.
  3. 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
GFP GFP Fluorescence End Point Normal 1 ### ### Bottom 100


Part 2: Measure background protein levels and normalize the fluorescence (IMPORTANT)

  1. 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.
  2. 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.
  3. In your figure (poster, paper, thesis, etc.) report the Fluorescence per μg protein.
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