Haynes:ExtFluorProtein

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Bradford Assay

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

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.

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

1. Follow the Bradford Assay procedure.
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 total protein.