ALL PHOSPHATES NEED A BLIGH/DYER EXTRACTION BEFORE THEY ARE READY FOR ANALYSIS
Bligh and Dyer Extraction (for Phosphate Analysis)
Reagents:
Chloroform:Methanol (1:2)
Distilled H2O, pH = 7.0
Chloroform purified through Aluminum Oxide
Procedure:
1. After JB Extraction Procedure*, aliquot 1.0 mL** of extract to glass screw-cap tubes.
2. Evaporate aliquots using nitrogen evaporator.
3. Add 3.0 mL of CHCl3:MeOH (1:2) to dried samples, vortex.
4. Add 0.8 mL of distilled H¬2O, vortex.
5. Add 1.0 mL Purified CHCl3, vortex.
6. Add 1.0 mL distilled H2O.
7. Cap tubes tightly, vortex very well.
8. Centrifuge tubes at 3000 RPM, 5 minutes, high brake.
9. Aspirate upper, aqueous phase (approx. 3.8 mL)
10. Aliquot lower, organic phase to new test tubes for Pi Analysis (in duplicates)
Usually: 2.0 mL Bligh & Dyer Extract
700 μL per tube X 2
Follow Protocol for Phosphate Assay
* Bligh and Dyer Extraction only needs to be done after Cell Removal from Plate and JB Extraction Procedure (Extraction Procedures vary depending if sample is Cell/Tissue or Yeast).
If following Cell Removal and Bligh & Dyer Extraction Procedure go straight to Phosphate Assay Protocol
** If Sphingomyelin Analysis is to be requested only aliquot .5ml of extract for Pi.