Fluorometry of solubilized cell extracts

Measuring Steady State Fluorescence Emission Spectra in Cell Lysates

Workflow

1. Prepare cell lysates.
2. Measure fluorescence emission spectra of the cell lysates.

Prepare Cell Lysates

 Equipment

1. Nutator
2. 4°C cold room
3. Centrifuge, such as a Beckman Optima TLX or Eppendorf microfuge.

Buffers and Reagents

1. Solubilization buffer(s).  We have used the two buffers below.
   1. 20 mM Tris pH 7.4, 100 mM NaCl, 1.0% n-dodecyl-b-D-maltoside (dodecyl maltoside, DDM).
   2. 50 mM Tris pH 7.8, 1.0% Nonidet P-40, 0.5% sodium deoxycholate, 150 mM NaCl, 5 mM EDTA.
   3. Solubilization buffers contain protease inhibitors at the following final concentrations: 0.5 mM PMSF, 5 µg/ml leupeptin, 1X Halt protease inhibitor cocktail (ThermoFisher Scientific 87786).
2. Pellets harvested from cells labeled with NAI-A594 or NAI-A488. Labeling is done with live cells in the growing media plus 30 nM of NAI-A594 or NAI-A488 for 30 min, 37°C, 5% CO₂.

Protocol

1. Lyse cell pellets in solubilization buffer.
   1. Typically, a cell pellet from one confluent 100 mm tissue culture dish will be lysed in 300 µl of solubilization buffer.
   2. The cell pellet is thoroughly resuspended by pipetting with a pipette-man and then the lysate is incubated with nutation for 0.5 to 1 h, 4°C.
2. Insoluble material is then removed from the lysate by centrifugation at 100,000 x g for 30 min or using Eppendorf microcentrifuge at 14,000 rpm, 20 min, 4°C.
3. The resulting supernatant (cell lysate) is collected and then either used immediately or stored at -80°C for later analysis.

Measure Steady State Fluorescence Emission 

Equipment

1. Fluorescence spectrometer (e.g. PTI Quanta Master)
2. Microcuvette (Starna)
   1. Recommended cuvettes: Starna 2 mm (3-2.45 or type 16-F style cuvettes).

Buffers and Reagents

1. Cell lysates prepared from NAI-A594 or NAI-A488 labeled cells.
2. Cell solubilization buffer.

Fluorometer Settings

1. Excitation slits are set to 1 nm.
2. Emission slits are set to 12 nm.

Emission Scan Settings

1. Excitation wavelength (e.g. 590 nm for Alexa 594 and 470 nm for Alexa 488)
2. Emission wavelengths (e.g. 600-700 nm for Alexa 594 and 485-600 nm for Alexa 488)
3. Length of scan is populated automatically
4. Step size: 1 nm
5. Integration time: 1 s
6. Averages: 1

Procedure

1. Place 70 ul sample in the cuvette (70 ul) and measure  fluorescence emission spectra.
2. First read cell solubilization buffer, which will give the background fluorescence of the buffer solution.
3. Remove the buffer solution from the cuvette by aspiration and then add 70 ul undiluted cell lysate and read fluorescence.

Data Analysis

1. Subtract the emission spectra of solubilization buffer from emission spectra measured in cell extracts.
2. Plot Fluorescence intensity vs. wavelength.