Method for specific identification of target biomolecules
The present invention relates to a method for identification of specific target proteins in a protein sample following a detection procedure, such as a Western blotting procedure, wherein the membrane is probed with at least two primary antibodies directed against the same and/or different epitopes of the same target protein, and wherein specific binding to the target protein in a sample is differentiated from unspecific binding to the target protein by comparing the resulting sample patterns, such as bands or spot patterns, with each other. In a further step signals from the true target proteins are enhanced while signals resulting from unspecific binding are diminished.
1. A method for identification of specific target biomolecules in a sample in a detection procedure, comprising:
probing a gel or membrane with at least two probes that are directed against and specifically bind to a same target biomolecule to form bands or spot patterns on the gel or membrane;
obtaining image sample patterns corresponding to the bands or spot patterns;
overlaying the image sample patterns with each other;
generating image data by applying an algorithm that uses pixel-wise multiplication to enhance overlapping features and diminish non-overlapping features of the bands or spot patterns in the overlayed image sample patterns;
scaling the image data using a root function; and
differentiating the bands or spot patterns corresponding to specific binding of the probes to the target biomolecule in the sample from non-specific binding of the probes to other biomolecules in the sample using the scaled image data.
2. The method of claim 1 , wherein the target biomolecule is a protein, a peptide, an aggregated biomolecule, a fusion protein, a modified protein, a phosphorylated protein, a glycosylated protein, an ubiquitinated protein, a SUMOylated protein, or an acetylated protein.
3. The method of claim 1 , wherein the scaling of the image data uses a function involving image data from original images.
4. The method of claim 1 , wherein the resulting sample pattern is quantitatively analyzed.
5. The method of claim 1 , further comprising:
pre-processing the sample; and
separating the sample by electrophoresis after the pre-processing step.
6. The method of claim 5 , wherein the pre-processing of the sample comprises affinity separation by using antibodies against the target biomolecule.
7. The method of claim 5 , wherein at least two images represent aliquots of a sample that have been covalently labeled with different dyes and mixed together during the pre-processing of the sample.
8. The method of claim 1 , wherein the detection procedure is a Western blotting procedure.
9. The method of claim 8 , wherein the image data is in a form of one or more intensity profiles or 3D surfaces.
10. The method of claim 8 , wherein the image data is in a form of one or more 3D surfaces.
11. The method of claim 1 , wherein a final result is the image data presented in a form of one or more images where color coding has been used to highlight the overlapping and/or non-overlapping features of the bands or spot patterns.
12. The method of claim 11 , wherein the color coding represents a result of applying the algorithm to the image data.
13. The method of claim 1 , wherein at least two primary antibodies are used as probes.
14. The method of claim 13 , wherein the at least two primary antibodies are from different species.
15. The method of claim 13 , wherein the at least two primary antibodies are from the same species.
16. The method of claim 13 , wherein the at least two primary antibodies are differentially labelled.
17. The method of claim 13 , wherein one or more of the antibodies are detected using Enhanced chemiluminescence (ECL).
18. The method of claim 13 , wherein the primary antibodies are labeled with fluorescent dyes.
19. The method of claim 13 , wherein differentially labelled secondary antibodies are used against the at least two primary antibodies.
20. The method of claim 19 , wherein the secondary antibodies are labeled with fluorescent dyes.
21. The method of claim 20 , wherein the fluorescent dyes are cyanine dyes.