I'm trying to find the concentration of liposomes by stewart assay. the assay works for phospholipids (lecitin) to draw a standard curve, but not for liposomal suspension. I think maybe liposomes were not solved in chloroform. do I need to make liposomal lysate before assay?
But people say it works for them!
The Steward assay is based in a liquid-liquid extraction (water/chloroform). Liposomes are destroyed when pass to the organic phase from the water suspension. In principle it works quite well. Check the concentration of your sample. If it is too low you will not detect the phospholipids (take also into account that the phospholipid polar head also modifies the standard curve because of their different distribution constants). Also, if the sample is too much concentrated, you can have problems because an interphase between water and chloroform can be originated.
Alternatively, and depending on the type of sample, you can carry out a phospholipid extraction using the monophase method (Folch et al). Once the organic phase is obtained it is rotovapored and the dry film dissolved in the appropriate volume of chloroform. Subsequently the Steward reactive is added and extraction carried out.
Remember, it is mandatory to use glass when you work with chloroform.
You can use the Bötcher et al. method (Bötcher, C. J. F., C. M. van Gent, and C. Fries. 1961. A rapid and sensitive sub-micro phosphorus determination. Anal. Chim. Acta. 24:203–204). This is the routine phosporus determination method used for the liposome concentration calculation in a sample.
Try it...
Ferrothiocyanate reagent: Prepare it by dissolving 2.703 g of ferric chloride
hexahydrate and 3.04 g of ammonium thiocyanate in double distilled water and make volume up to 100 mL. Prepare Phopholipid stock (100 μg/mL) by dissolving 10 mg phosphotidylcholine in 100 mL of chloroform.
Method: Mix Solutions (2 mL) of different concentrations (10, 20, 40, 60, 80 μg/mL) with 2 mL of ferrothiocyanate reagent followed by vortexing for 20 sec and centrifugation at 1000 rpm. Remove the lower chloroform layer using Pasteur pipette and read optical density at 485 nm.
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