Assays for detecting modified compounds
Provided are methods and compositions which are useful for separating, isolating, detecting, and quantifying compounds of interest which have been modified chemically, enzymatically or catalytically from other compounds which have not been so modified. The modifications may take the form of functional groups which are gained, lost or retained by the compounds of interest.
1. A method for detecting or quantifying an enzyme capable of adding a functional group to a compound in a sample, the method comprising the steps of:
(a) providing:
(i) a compound which can gain a functional group from an enzyme, the compound being directly covalently bound to at least one non-radioactive signaling moiety and the compound comprising sphingosine;
(ii) a sample suspected of containing an enzyme that could add a functional group to the compound; and
(iii) a chemical, enzymatic or catalytic source of the functional group which can be gained by the compound; and
(iv) means to separate the compound which has gained the functional group from the compound which has not gained the functional group;
(b) forming a mixture comprising the compound, the sample and the source of the functional group, and incubating the mixture under conditions suitable for the compound to gain the functional group;
(c) separating and thereby isolating any compound which has gained the functional group from any compound which has not gained the functional group by precipitating said compound which has gained the functional group from the mixture by the addition of a charged metal ion and a solvent selected from the group consisting of ethanol, methanol, isopropyl alcohol, dioxane, and combinations thereof;
(d) resolubilizing said compound which has gained the functional group;
wherein the functional group comprises a phosphate.
2. The method of claim 1 , further comprising detecting or quantifying the non-radioactive signaling moiety in said resolubilized compound having the functional group, to detect or quantify any compound which has gained the functional group.
3. The method of claim 1 , wherein the chemical, enzymatic or catalytic source of the functional group comprises an enzyme and a chemical comprising the functional group.
4. The method of claim 1 , further comprising providing a normalizing reagent to promote precipitation of the compound that has gained the functional group.
5. The method of claim 1 , wherein barium acetate and ethanol are added for precipitation; wherein EDTA is used for resolubilizing the compound and wherein the non-radioactive signaling moiety comprises a fluorescent compound, a phosphorescent compound, a chemiluminescent compound, a chromogenic compound, a chelating compound, an electron dense compound, a magnetic compound, an energy transfer member or pair, an intercalating compound, or any combination thereof.
6. The method of claim 1 , further comprising blocking at least one moiety on the compound that interferes with the compound gaining the functional group in step (b) or separating the compound in step (c).
7. The method of claim 2 , wherein the detecting or quantifying is carried out directly or indirectly.