Composition and method for gel electrophoresis with in-situ calibration
The invention relates to, among other things, a method for performing electrophoresis with in-situ calibration. The method includes combining a volume of a test sample with a volume or quantity of a calibrating sample to form a final volume, where the volume of the calibrating sample includes a known concentration of a calibrator and the final volume includes a known ratio of test sample to calibrating sample. The method also includes depositing a loading fraction in a receiving well of an electrophoretic gel, in which the loading fraction is a fraction of the final volume, and separating the loading fraction along a common separation lane of the electrophoretic gel such that components of the test sample and the calibrator are separated from one another along the common separation lane. The method also includes detecting the calibrator and separated components of the test sample within the common separation lane and measuring the level of the calibrator and separated components of the test sample based on the detecting, thereby performing electrophoresis with in-situ calibration.
1. A method for performing polyacrylamide or agarose gel electrophoresis with in-situ calibration, the method comprising:
combining a volume of a test sample with a volume of a calibrating sample to form a final volume, wherein the volume of the calibrating sample comprises a known concentration of a calibrator and the final volume comprises a known volumetric ratio of test sample to calibrating sample;
depositing a loading fraction in a receiving well of a polyacrylamide or agarose electrophoretic gel, wherein the loading fraction is a fraction of said final volume;
separating the loading fraction along a common separation lane of the polyacrylamide or agarose electrophoretic gel such that at least one protein component of the test sample and at least one protein component of the calibrator are separated from one another along the common separation lane;
detecting the at least one protein component of the test sample and the at least one protein component of the calibrator within the common separation lane;
wherein said one or more protein components of the test sample comprises lipoprotein particles or portions thereof and wherein said detecting comprises detecting the lipoprotein particles or portions thereof, and
quantitating the level of the at least one protein component of the test sample and the at least one protein component of the calibrator based on said detecting, thereby performing electrophoresis with in-situ calibration.
2. The method according to claim 1 , further comprising: calculating the concentration of the at least one protein component of the test sample in said final volume based on the measured level of the at least one protein component of the calibrator, the measured level of the at least one protein component of the test sample, and the known volumetric ratio of test sample to calibrating sample.
3. The method according to claim 1 , wherein the at least one protein component of the test sample and the at least one protein component calibrator are each bound to a signal producing molecule capable of producing or causing production of a detectable signal.
4. The method according to claim 3 , wherein the method further comprises: contacting the separated protein components of the test sample and/or the calibrator with a reagent capable of interacting with the signal producing molecule, wherein the signal producing molecule produces the detectable signal upon contact with the reagent and wherein said detecting comprises detecting the detectable signal.
5. The method according to claim 3 , wherein the protein components of the test sample and the calibrator are bound to signal producing molecules that are distinguishable from one another.
6. The method according to claim 3 , wherein the protein components of the test sample comprise at least two different protein components.
7. The method according to claim 6 , wherein each of the at least two protein components is bound to a different detectable signal, each of the detectable signals being distinguishable from one another.
8. The method according to claim 3 , wherein the detectable signal is detectable by radiometric, colorimetric, luminometric, or fluorometric means.
9. The method according to claim 1 , wherein the lipoprotein particles or portions thereof are selected from the group consisting of Apolipoprotein A, Apolipoprotein B, Apolipoprotein C, Apolipoprotein D, Apolipoprotein E, Apolipoprotein H, lipoprotein (a), high density lipoprotein, intermediate density lipoprotein, low density lipoprotein, very low density lipoprotein, Chylomicrons, Lipoprotein X, oxidized variants and mixtures thereof.
10. The method according to claim 1 , wherein said depositing is carried out with an electrophoretic comb.
11. The method according to claim 1 , wherein said calibrator is a fluorophore.
12. The method according to claim 1 , wherein the calibrator comprises albumin.
13. The method according to claim 12 , wherein the albumin is coupled to a signal producing molecule.
14. The method according to claim 12 , wherein the albumin is coupled to a fluorophore.
15. The method according to claim 1 , wherein said calibrator migrates on an electrophoretic gel during gel electrophoresis at a faster rate than components of the test sample.
16. The method according to claim 9 , wherein said calibrator migrates on an electrophoretic gel during gel electrophoresis at a faster rate than the lipoprotein particles.
17. The method according to claim 1 , wherein the test sample is a biologic sample.
18. The method according to claim 1 , wherein the calibrator and the separated protein components of the test sample are fixed prior to said detecting.
19. The method according to claim 18 , wherein the calibrator and separated components of the test sample are fixed by an immunofixation technique, the immunofixation technique comprising:
contacting the calibrator and separated components of the test sample with antisera comprising a first antibody or fragment thereof and a second antibody or fragment thereof, such that the first antibody or fragment thereof binds the calibrator and the second antibody or fragment thereof binds the separated component of the test sample; and
washing the electrophoretic gel to remove unbound materials in the gel.
20. The method according to claim 1 , wherein said calibrator comprises a protein or polypeptide bound to a fluorophore.