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Comparison of Classical P81 and SVI-P Cation-Exchange Papers in Radiometric Protein Kinase Assays.

Muhammad Khabib^1,2, Lifang Zhang¹, Ashley Ovens²

¹Metabolic Signalling Laboratory, St Vincent's Institute of Medical Research, Fitzroy, Victoria, Australia, CA.

The Biochemical Journal

|July 23, 2025

Summary

A new filter paper, SVI-P, effectively replaces P81 for radiometric kinase assays, ensuring reliable detection of phosphorylated substrates in biochemical research.

Keywords:

Biochemical techniques and resources P81 SVI-P [γ-32P]-ATP kinases radiometric kinase assay

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Area of Science:

Biochemistry
Molecular Biology
Enzymology

Background:

Radiometric kinase assays are sensitive methods for studying enzyme activity.
These assays rely on phosphocellulose paper (P81) for capturing phosphorylated substrates.
The discontinuation of P81 supply necessitates an alternative filter paper.

Purpose of the Study:

To evaluate SVI-P cation-exchange filter paper as a replacement for P81 in radiometric kinase assays.
To assess the performance of SVI-P across various kinase assays and substrate types.

Main Methods:

Radiometric kinase assays were performed using SVI-P and P81 filter papers.
Substrate capture efficiency was compared between the two filter types.
Kinase profiling assays were conducted using SVI-P with diverse protein kinases.

Main Results:

SVI-P demonstrated comparable substrate capture efficiency to P81 in assays for AMPK, Abl, CDK2, and ERK.
SVI-P successfully captured peptide and protein substrates in a commercial kinase profiling assay involving 48 kinases.
Higher background radioactivity was observed with SVI-P, but manageable through optimization.

Conclusions:

SVI-P is a viable and effective alternative to P81 for radiometric kinase assays.
This replacement ensures the continuity of kinase research in academic and industrial settings.
Optimization of wash steps can mitigate the higher background radioactivity associated with SVI-P.