Kits to assay kinase activity
Methods and kits for enzymes involved in post-translational modifications are provided. The methods employ elemental analysis, including ICP-MS. The methods allow for the convenient and accurate analysis of post-translation modifications of substrates by enzymes involved in post-translational modifications, including kinase and phosphatase enzymes. Kits may include an element tag for directly tagging an affinity product that recognizes phosphorylated substrates, as well as a non-phosphorylated substrate or an expression plasmid.
1. A kit for detecting and measuring elements in a sample, where the measured elements include element tags attached to affinity products that recognize phosphorylated substrates, comprising:
a) non-phosphorylated substrate ready to be introduced into a cell; and
b) an element tag for directly tagging the affinity products that recognize phosphorylated substrates.
2. The kit of claim 1 further comprising instructions for i) introducing the non-phosphorylated substrate into the cell; ii) directly tagging the affinity product that recognizes phosphorylated substrates with the element tag; iii) fixing and permeabilizing the cell; iv) combining the labeled affinity product with the cell; v) separating bound affinity product from unbound affinity product, and vi) detecting and measuring the amount of the bound affinity product labeled with the element tag by particle elemental analysis.
3. The kit of claim 1 further comprising a multitude of non-phosphorylated substrates to be introduced into the cell.
4. The kit of claim 1 wherein the non-phosphorylated substrate is in a sterile solution at a concentration compatible with microinjection into the cell.
5. The kit of claim 1 further comprising an affinity product that recognizes phosphorylated substrates, wherein the affinity product is directly labeled with the element tag.
6. The kit of claim 1 further comprising a different types of affinity products that recognize phosphorylated substrates, wherein the different types of affinity products are directly labeled with element tags that are unique to the type of affinity product.
7. The kit of claim 1 further comprising an expression plasmid and wherein the non-phosphorylated substrate is produced by an expression plasmid transfected or electroporated into the cell.
8. The kit of claim 1 wherein the non-phosphorylated substrate is encapsulated in a lipid microsome.
9. The kit of claim 1 further comprising the affinity product that recognizes the phosphorylated substrates and wherein the affinity product that recognizes the phosphorylated substrates is selected from the group consisting of antibody, Fab′, aptamer, antigen, hormone, growth factor, receptor, protein, peptide, SH2 peptide, and nucleic acid.
10. The kit of claim 1 , wherein the non-phosphorylated substrate is element tagged.
11. The kit of claim 1 , wherein the non-phosphorylated substrate is not element tagged.
12. The kit of claim 1 , wherein the non-phosphorylated substrate is conjugated with a protein transfer domain sequence which enables the non-phosphorylated substrate to be taken up into a cytoplasm of the cell.
13. A kit for detecting and measuring f elements in a sample, where the measured elements include element tags attached to affinity products that recognize phosphorylated substrates, comprising:
an expression plasmid configured to be transfected or electroporated into a cell, wherein a non-phosphorylated substrate is produced by transfected or electroporated expression plasmid; and
an element tag for directly tagging the affinity products that recognize phosphorylated substrates.
14. The kit of claim 13 further comprising an affinity product that recognizes phosphorylated substrates, wherein the affinity product is directly labeled with the element tag.
15. The kit of claim 14 wherein the affinity product that recognizes the phosphorylated substrates is selected from the group consisting of antibody, Fab′, aptamer, antigen, hormone, growth factor, receptor, protein, peptide, SH2 peptide, and nucleic acid.