Phosphate Uptake Assay

Categorized: Outer MembranesBiochemical Assays

Reference:

Poole and Hancock. (1848.) Eur. J. Biochem. 144:607-612.

Method:

  1. Purchase 32P, phosphoric acid in HCl 2.0 mCi/ml from NEN (NEX-054).
  2. Set up 10-25 ml overnight cultures in low phosphate media (see recipe file for Pseudomonas aeruginosa and E. coli low phosphate Hepes buffered minimal media). Harvest cells by filtration with Nalgene 0.45 µm filter units. wash cells by filtration 3X with 10 ms of Hepes buffered minimal media WITHOUT phosphate.
  3. Resuspend washed bacteria in the same phosphate-free media to a final OD600 = 0.2-0.3. Store on ice until needed. Cells are good for about 3 hours.
  4. Place 32P (2.5 µM = 1.25 µl of source) in a 10 ml diposable tube.
  5. Warm bacterial suspension approximately 10 minutes at 37ºC before using in assay. Take one ml sample of bacterial suspension and add to the 32P. Immediately start timing, vortex to insure good mixing. Remove 200 µl samples at the desired time intervals and filter using 0.45 nitrocellulose filters. (The old manifold filtration apparatus with filter cups from Amicon is useful for this.) Immediately wash the filter with Hepes buffered minimal media WITH 1 mM Phosphate (from a squirt bottle for fast washing). For a good time course try 15, 30, 45, 60 seconds after mixing cells with 32P.
  6. Remove filters , dry and count in 5 ml of scintillation cocktail.
  7. Duplicate experiments should be done on two different days to verify results.
  8. Controls: 200 µl of Hepes buffered media alone to get background counts and 1.25 µl of 32P in 1 ml buffer, no cells, to get maximum counts.