Assay of phycobiliproteins in Fremyella diplosiphon

This protocol was graciously provided by: John Cobley, Dept of Chemistry, University of San Francisco, U.S.A. Cobley@Usfca.Edu

The advantage of this method is that the resulting supernatant is totally free of chlorophyll which interferes with biliprotein determination. If you sonicate cells then it is hard to spin down all of the chlorophyll. This method gives quantitative recovery of biliproteins since cell lysis is complete as viewed under the microscope. (F. diplosiphon cells are very easy to lyse).

REAGENTS

Solution I 0.01M Na2HPO4, 0.15M NaCl, pH 7.0 Solution II O.5% (W/V) lysozyme in Solution I

PROCEDURE

1. Centrifuge the culture in a autoclaved 50ml Corning Orange Cap tube in the International centrifuge at 2,500 rpm for 15 min.

2. Transfer the pellet to a 1.5ml microtube and microfuge for 10 min.

3. Pour off the supernatant, and dry the pellet with the twisted corner of a Kimwipe. Freeze the pellet at -20deg C.

4. Thaw and resuspend in 1.25ml of Solution I.

5. Add 150ul of 0.5% lysozyme (Solution II). Vortex.

6. Incubate at 37deg C for 2h with reciprocal shaking in a water bath.

7. Stand the tubes O/N at 4deg C.

8. Mix contents of tube by briefly vortexing.

9. Spin in the microfuge for 30 min.

10. Remove 1ml from the supernatant with a P1000 Pipetman. Avoid contamination with the pellet. Transfer this 1ml to a tube of at least 2ml capacity.

11. Bring to 2ml by adding 1ml of Solution I.

12. Scan solution from 750-500 nm using the Perkin Elmer 330 Spectrophotometer. Dilute if necessary using Solution I.

ALTERNATIVE PROCEDURE FOR WHEN THE NUMBER OF PACKED CELLS IS SMALL

4 (alt). Use only 250 ul of Solution I

5 (alt). Use only 30 ul of lyzozyme solution.

10 (alt). Remove only 200 ul of the supernatant, and scan in the black-walled microcells with 0.5 cm path length.

REFERENCE

BENNETT, A., and BOGORAD, L. (1973). Complementary Chromatic Adaptaption in a Filamentous Blue-Green Alga. J. Cell Biol. 58, 419-435