Amino Acid Transport Assays in Resting Cells of Lactococcus lactis

Materials and Reagents

1. L. lactis cells
   
2. Whatman, Protran^® BA85 Nitrocellulose Membranes (2.5 cm diameter, 0.45-μm-pore-size nitrocellulose filter ) (Sigma-Aldrich, catalog number: Z670634 )
   
3. LiCl
   
4. K₂HPO₄
5. KH₂PO₄ 
   
6. ¹⁴C-labelled amino acids (PerkinElmer)
   Note: stock solutions typically contain 50-100 mCi/ml corresponding to 100 to 500 μM concentrations.
   
7. Scintillation fluid (e.g. Emulsifier Scintillator Plus, Packard Bioscience)
8. 100 mM potassium phosphate (KPi) pH 5.8 buffer at 4 °C (see Recipes)
9. KPi containing 0.2% glucose at 4 °C (see Recipes)
   

Equipment

1. Filtration setup (e.g. Ten-place filtration manifold, catalog number: FH225V )
   
2. Vacuum pump
   
3. Thermostat water bath with magnetic stirrer
   
4. Liquid scintillation analyser (e.g. Tri-Carb 2000CA, Packard Instruments)
   
5. Glass tubes (10 x 75 mm)
   
6. Tweezers
   
7. Magnetic stirring bars
   
8. Timer
   
9. Vortex

Procedure