A versatile polyacrylamide gel electrophoresis based sulfotransferase assay
Zhengliang L Wu1, Cheryl M Ethen, Sara Larson
1R&D Systems Inc, 614 McKinley Place NE, Minneapolis, MN 55413, USA. leon.wu@rndsystems.com
BMC Biotechnology
|February 12, 2010
Summary
A new universal sulfotransferase assay using SDS-PAGE simplifies enzyme analysis. This method works for various substrates and can be adapted for other enzymes like kinases.
Area of Science:
- Biochemistry
- Enzymology
Background:
- Sulfotransferases are crucial enzymes involved in sulfation.
- Assaying sulfotransferases is challenging, necessitating improved methods.
- Understanding these enzymes requires convenient and versatile assays.
Purpose of the Study:
- To develop a universal assay for sulfotransferase enzymes.
- To establish a method applicable to a wide range of substrate sizes.
- To provide a tool for better understanding sulfotransferase function.
Main Methods:
- Utilized sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for a universal assay.
- Applied the method to diverse substrates, from small molecules like alpha-naphthol to large proteoglycans.
- Developed a predictive method for small molecule applicability based on relative mobility to PAPS.
Main Results:
- Successfully demonstrated the assay for multiple sulfotransferases (CHST4, TPST1, CHST3, HS6ST1).
- The assay accommodates substrates of varying molecular weights.
- Predicted relative mobilities correlated well with actual mobilities for sulfated small molecules.
Conclusions:
- SDS-PAGE's differential separation of proteins and small molecules enables versatility.
- The method's applicability is indicated by the predicted relative mobility of products to PAPS.
- The assay is potentially adaptable for kinase assays due to chemical similarities with sulfation.
Related Concept Videos
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...
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.
Biological samples, such as cells...
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.
Biological samples, such as cells...