Uromodulin (THP) can be a difficult protein to work with because it self-aggregates affecting the quality of downstream applications.  The degree of aggregation can depend on the pH and ionic strength of urine at the time of collection.  This reference guide summarizes a few techniques from the literature to optimize THP for various assays.

Sample collection and storage:

Urine samples should be aseptically collected in mid stream and directly into a sterile container. It is recommended that if the samples can’t be used immediately, that the undiluted samples should be stored at -20 ºC. Aliquots are recommended to avoid repeated freeze-thaw cycles. 

Sample preparation:

The availability of Uromodulin in the urine sample may be affected by pH and the ionic strength of the urine sample. Published data have shown that correct pH and ionic conditions are required to avoid loss of the analyte through aggregation and subsequent sedimentation. The MD-2 Assay Diluent provided in this kit is designed to keep the Uromodulin in soluble form providing better linearity of the sample results. Other recommended buffers include the TEA (Triton/EDTA/Alkaline) Buffer containing 0.5% Triton x-100, and 20 mM EDTA at a pH of 7.5 .

To remove particulate matter from the urine, the samples after dilution with MD-2 Assay Diluent or TEA Buffer can be centrifuged at low speed at 400x g for 5 minutes.

Sample Dilution: 

Uromodulin levels in urine may be highly variable and appropriate dilutions must be determined by each individual lab. For human urine samples, a suggested starting dilution of 1:200 is recommended.