I am making carbamylated LDL, and need to run the homocitrulline assay to verify that the end product works. Could anyone kindly provide me with a protocol to the homocit assay for this purpose
Thanks
Dear Ronny,
The following text contains the requested protocol:
A colorimetric method using diacetyl monoxime11 was used to measure the degree of carbamylation in LDL preparations. Briefly, the LDL suspension (25 g of protein) was digested in 50 L phosphate-buffered saline (PBS), pH 7.4, 1% sodium dodecyl sulfate (SDS) with 2 g proteinase K at 37°C for 2 hours. Then 250 L of urea-nitrogen reagent (0.83 mol/L sulfuric acid, 1.13 mol/L orthophosphoric acid, 0.55 mmol/L thiosemicarbazide, and 2.6 mmol/L cadmium sulfate) and 50 L diacetyl monoxime were added to the reaction mixture and the incubation continued at 97°C for 30 minutes. Precipitate was removed by centrifugation at 3500g for 10 minutes at room temperature. The supernatant (200 L) was transferred into a 96-well plate and absorption was measured at 530 nm. A standard curve was generated using homocitrulline (e-amino-carbamyllysine, 0 to 30 nmol) (Advanced Asymmetrics, Millstadt, IL, USA). The results were expressed in nmol homocitrulline/mg LDL protein.
For more details, use the following link:
http://www.nature.com/ki/journal/v68/n1/full/4496075a.htm
Hoping this will be helpful,
Rafik
Here's another paper on the subject:
http://www.ncbi.nlm.nih.gov/pubmed/22160237
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