I need to determine concentration of heparin in sample with the method described by Frazier et al, but it doesn't work.. do you have any tips?
Thanks!
Dear Allison,
I suggest to read the following text which describes the procedure for the determination of heparin. The text contains several hints which may be helpful:
Detection of charge-neutralizing factors
As the only initially predicted property of the charge-neutralizing factor was that it would bind to and neutralize the negative charge on cells and membranes, we made use of this property for its detection. The anionic GAGs heparin, heparan sulphate, chondroitin sulphate, and dermatan sulphate are among the most anionic constituents of biological membranes [27, 28], and are also present in plasma [291 and urine [30]. Most methods for detection of GAGs utilize their negative charge and involve precipitation of GAGs from solution by cationic dyes or detergents [30—32]. We used the method of Whiteman [30, 33], in which the cationic dye Alcian blue 8GX is used to precipitate GAGs from plasma and urine. On mixing Alcian blue with GAGs in solution, a precipitate is formed which at pH 5.8 in the presence of magnesium chloride is specific for GAGs [30, 331. The Alcian blue in the precipitate can be quantified after solubilization in SDS, by measurement of the optical density at 678 nm. The concentration of GAGs in the original solution is then calculated by reference to the Alcian blue precipitate formed using a standard GAG solution. Although this method is largely unaffected by normal plasma or urine proteins [30, 33], highly cationic proteins such as protamine or polylysine, which bind to GAGs with high affinity, would be expected to resist displacement by Alcian blue. The
presence of protamine or protamine-like cationic factors might, therefore, be inferred by their ability to inhibit the binding of Alcian blue to GAGs. To confirm that highly cationic proteins could be detected by their ability to interfere with the binding of Alcian blue to GAGs, the amount of Alcian blue precipitated was determined for standard GAG solutions (heparin 0 to 40 U/ml in distilled water) in the presence and absence of protamine sulphate 2 mg/ml. One hundred microliters of the heparin solution or the heparin/protamine mixture was added to 2 ml Alcian blue solution (0.05% wt/vol Alcian blue 8GX in 50 m sodium acetate buffer pH 5.8 containing 50 ma MgCI2). After incubation at room temperature for three hours, the precipitate was recovered by centrifugation at 2,000 g for 15 minutes. The precipitate was washed once in 2 ml ethanol and again centrifuged. After decantation of the ethanol, the precipitate was dissolved in 1 ml 7.5% (wt/vol) sodium dodecyl sulphate (SDS) and the optical density (OD) at 678 nm recorded. The resulting standard curve of the Alcian blue precipitate obtained with different concentrations of heparin, in the presence or absence of protamine was included as a control in all subsequent experiments (Fig. 1).
For more information, please see Methods section in the publication contained in the following link:
http://ac.els-cdn.com/S0085253815347141/1-s2.0-S0085253815347141-main.pdf?_tid=34188764-8c17-11e6-955c-00000aacb35f&acdnat=1475794377_7d1a9dc51b7a7097de91daf59d96fe36
Hoping this will be helpful,
Rafik
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