The original Stewart assay protocol was published by J.C.M Stewart in Journal of Analytical Biochemistry in 1980:
Stewart, J.C.M. (1980). Anal Biochem, 104, 10.
Based on the request of several scientists we have posted the Stewart assay method in this blog entry.
Stewart assay is based on the ability of phospholipids to form a complex with ammonium ferrothiocyanate.
The advantage of this method is that unlike Bartlett assay, the presence of inorganic phosphate does not interfere with this assay and therefore PBS buffer or other phosphate based buffers can be used.
A simple conversion factor is used to translate the absorbance values (from the spectrophotometer) into milligrams of phospholipid. Keep in mind that this conversion factor is different for phospholipids with different head groups, therefore this method is not applicable to samples where mixtures of unknown phospholipids may be present.
One of the drawbacks of this method is its inability to quantify phosphatidylglycerol(PG).
Equipment and reagents
1) 10 ml centrifuge tubes
Preparation of reagents and standards
Ferrothiocyanate Reagent: Dissolve 27.03 gram of ferric chloride hexahydrate (Sigma Aldrich Cat. no. F2877) and 30.4 gram of ammonium thiocyanate (Sigma Aldrich Cat. no. 221988) in double distilled water and volumized it to 1 liter. The solution is stable at room temperature for several months.
Standard: Make up 10 ml of solution of phospholipid in chloroform at concentration of 0.1 mg/ml
1) Pipette reagents and standard into the centrifuge tubes as follows:
Prepare triplicates of each tube. Perform the same procedure with the test samples.
2) Vortex each tube for 20 seconds.
3) Spin each tube for 10 minutes at 1000 r.p.m (approx 300 g) in centrifuge and remove the lower layer using a Pasteur pipet.
4) Read the optical density of standards and samples at 485 nm.
5) Find the concentration in the test sample by comparing it with the standard curve.
For Bartlett assay see here: