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Oligopeptide Competition Assay for Phosphorylation Site Determination
Published on: May 18, 2017
In-gel phosphatase assay using non-denaturing two-dimensional electrophoresis
Hiromi Baba1, Yukihiro Masuda, Noriyuki Sueyoshi
1Faculty of Agriculture, Department of Life Sciences, Kagawa University, Kagawa 761-0795, Japan.
Journal of Biochemistry
|September 21, 2012
Summary
This study introduces a novel two-dimensional electrophoresis method for detecting phosphatase activities in tissue extracts. The technique effectively visualizes diverse enzyme activities using a fluorogenic substrate after protein separation.
Area of Science:
- Biochemistry
- Molecular Biology
- Proteomics
Background:
- Phosphatases are crucial enzymes involved in various cellular processes.
- Analyzing phosphatase activity in complex biological samples like tissue extracts presents challenges.
- Existing methods may require protein denaturation or lack comprehensive activity profiling.
Purpose of the Study:
- To develop and validate a novel non-denaturing two-dimensional electrophoresis method for detecting phosphatase activities.
- To enable the analysis of diverse phosphatase activities in crude tissue extracts.
- To allow for sequential detection of enzyme activities under different pH conditions.
Main Methods:
- Proteins were separated using a novel non-denaturing two-dimensional electrophoresis.
- First dimension: liquid-phase isoelectric focusing (MicroRotofor) with or without urea.
- Second dimension: native polyacrylamide gel electrophoresis with 2-mercaptoethanol, followed by incubation with 4-methylumbelliferyl phosphate (MUP) and sequential pH analysis.
Main Results:
- The method successfully separated proteins and detected various phosphatase activities as fluorescent bands.
- Optimal detection of diverse phosphatase activities was achieved when tissue extracts were separated using MicroRotofor with 1.5 M urea.
- Sequential detection of phosphatase activities at neutral and acidic (pH 3.0) conditions was demonstrated.
Conclusions:
- The developed non-denaturing two-dimensional electrophoresis method is highly effective for analyzing phosphatase activities in crude tissue extracts.
- This technique allows for comprehensive profiling of multiple phosphatase activities under varying conditions.
- The method offers a valuable tool for biochemical and proteomic research involving enzyme activity analysis.
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01:22Two-dimensional Gel Electrophoresis
Two-dimensional gel electrophoresis is a high-resolution protein separation method first introduced by O' Farrell and Klose in 1975. This method involves protein separation by two dimensions, mass and charge, making it more accurate than one-dimensional gel electrophoresis.
The first dimension separation uses the isoelectric focusing or IEF technique performed on immobilized pH gradient (IPG) strips that separate proteins according to their isoelectric points.
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The first dimension separation uses the isoelectric focusing or IEF technique performed on immobilized pH gradient (IPG) strips that separate proteins according to their isoelectric points.
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01:27SDS-PAGE
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...
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...