Phosphate-affinity Sds-page
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08:55
Rab10 Phosphorylation Detection by LRRK2 Activity Using SDS-PAGE with a Phosphate-binding Tag
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Cited by 7 •
2017
The present study describes a simple method of detecting endogenous levels of Rab10 phosphorylation by leucine-rich repeat kinase 2.
07:29
Separating Protein with SDS-PAGE
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2023
Sodium Dodecyl Sulfate Poly-Acrylamide Gel Electrophoresis, or SDS-PAGE, is a widely-used technique for separating mixtures of proteins based on their size and nothing else. SDS, an anionic detergent, is used to produce an even charge across the length of proteins that have been linearized. By first loading them into a gel made of polyacrylamide and then applying an electric field to the gel, SDS-coated proteins are then separated. The electric field acts as the driving force, drawing the SDS...
01:27
SDS-PAGE
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2023
Gel electrophoresis is a method that separates biological macromolecules like nucleic acids or proteins by forcing them to pass through a gel matrix under an electric field. A variation of gel electrophoresis, termed polyacrylamide gel electrophoresis (PAGE), is commonly used for separating proteins according to their molecular size by passing them through a polyacrylamide gel. Because of the varying charges associated with amino acid side chains, PAGE can be used to separate intact proteins...
11:31
Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate
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Cited by 5 •
2013
Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are multidomain proteins which encode both GTPase and kinase domains and which are phosphorylated in cells. Here, we present a protocol to label LRRK1 and LRRK2 in cells with 32P orthophosphate, thereby providing a means to measure their overall cellular phophorylation levels.
03:07
Electron Affinity
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2020
The electron affinity (EA) is the energy change for adding an electron to a gaseous atom to form an anion (negative ion). This process can be either endothermic or exothermic, depending on the element. Many of these elements have negative values of EA, which means that energy is released when the gaseous atom accepts an electron. However, for some elements, energy is required for the atom to become negatively charged, and the value of their EA is positive. Just as with ionization energy,...
FAQs
The phosphate-binding reagent coordinates divalent metal ions to form a complex that transiently captures phosphorylated residues on proteins. These temporary interactions add resistance to migration through the polyacrylamide matrix, so phosphorylated species move more slowly than comparable nonphosphorylated species. The resulting mobility difference allows phosphorylation-dependent forms to be distinguished during electrophoretic analysis.
Multiple bands or mobility positions can indicate that a protein population contains forms with different phosphorylation states. Because phosphorylation can produce distinct shifts, the pattern may reflect differences in phosphorylation stoichiometry rather than differences in protein identity. Comparing these bands helps assess whether a sample contains nonphosphorylated, singly phosphorylated, or more extensively modified forms when those states are resolved.
Conventional SDS-PAGE primarily separates denatured proteins according to molecular size, whereas the phosphate-affinity format adds a phosphorylation-sensitive contribution to migration. The modified gel therefore can distinguish forms of the same protein that would otherwise have similar size and potentially overlap. This makes phosphorylation state an additional source of electrophoretic resolution rather than relying on size alone.
Mobility reflects both the protein's molecular size and its phosphorylation state. Size influences movement through the polyacrylamide matrix, while phosphorylated residues provide binding sites for the metal-coordinated phosphate-binding reagent. The combined effects determine whether related protein forms separate into discrete bands and whether differences in phosphorylation produce measurable mobility shifts.
The workflow uses a polyacrylamide gel containing a phosphate-binding reagent, such as Phos-tag, coordinated with divalent metal ions. Protein samples are separated electrophoretically through this modified matrix, and the resulting band positions are compared. Differences in migration are then interpreted alongside molecular-size effects to identify distinct phosphorylation-dependent forms.
This approach is useful when researchers need to monitor protein phosphorylation without requiring radiolabeling. It can help characterize kinase activity by tracking phosphorylation-associated mobility changes, evaluate phosphatase activity through altered band patterns, and follow regulation within signaling pathways. The method therefore connects electrophoretic protein analysis with biochemical studies of reversible phosphorylation and cellular signaling.