Assaying Protein Kinase Activity with Radiolabeled ATP

Aroon S. Karra; Steve Stippec; Melanie H. Cobb

doi:10.3791/55504

JoVE Journal
Biochemistry

A subscription to JoVE is required to view this content. Sign in or start your free trial.

JoVE Journal Biochemistry

Assaying Protein Kinase Activity with Radiolabeled ATP

방사성 표지 된 ATP로 단백질 키나아제 활성 측정

Full Text

19,464 Views

08:05 min

May 26, 2017

DOI: 10.3791/55504-v

Aroon S. Karra¹, Steve Stippec¹, Melanie H. Cobb¹

¹Department of Pharmacology,University of Texas Southwestern Medical Center

Please note that some of the translations on this page are AI generated. Click here for the English version.

Overview

This article presents a protocol for measuring the enzymatic activity of protein kinases using radiolabeled ATP. The method is versatile and quantitative, aiding in the understanding of kinase signaling.

Key Study Components

Area of Science

Biochemistry
Cell Biology
Signal Transduction

Background

Protein kinases are essential for cellular signaling.
Understanding kinase activity is crucial for various biological processes.
This assay can determine kinase specificity and activity under different conditions.
It utilizes radiolabeled ATP for accurate measurement.

Purpose of Study

To measure the activity of protein kinases.
To assess the effects of different treatments on kinase activity.
To evaluate the specificity of kinases and their mutants.

Methods Used

Incubation of cell lysates with antibodies.
Use of protein A-Sepharose beads for purification.
Quantitative measurement of kinase activity.
Application of radiolabeled ATP in the assay.

Main Results

The assay provides reliable measurements of kinase activity.
It demonstrates versatility across different experimental conditions.
Results can inform on kinase specificity and response to treatments.
Expert demonstration by a seasoned researcher enhances credibility.

Conclusions

This protocol is a valuable tool for studying protein kinases.
It contributes to the understanding of cellular signaling pathways.
Future applications may expand to various kinase-related research areas.

Frequently Asked Questions

What is the main goal of the assay?

The main goal is to measure the enzymatic activity of protein kinases.

Who demonstrates the procedure?

Steve Stippec, a research scientist at the Cobb Laboratory, demonstrates the procedure.

What is the advantage of this technique?

The technique is versatile and quantitative, allowing for detailed analysis of kinase activity.

How is the assay performed?

It involves incubating cell lysates with antibodies and using protein A-Sepharose beads for purification.

What type of ATP is used in the assay?

Radiolabeled adenosine triphosphate ([γ-32 P] ATP) is used for measuring kinase activity.

What can this assay help determine?

It can help determine the activity level of kinases and their specificity under various conditions.

단백질 키나아제는 고도의 진화 된 신호 효소 및 세포 내 및 세포 내 신호 전달에 중요한 발판이다. 우리는 세포 표지 조절의 해명에 도움이되는 신뢰할 수있는 방법 인 방사성 표지 된 아데노신 3 인산염 ([γ- ³² P] ATP)의 사용을 통해 키나아제 활성을 측정하기위한 프로토콜을 제시한다.

이 분석의 전반적인 목표는 단백질 키나아제의 효소 활성을 측정하는 것입니다. 이 방법은 키나아제 또는 키나아제 돌연변이가 얼마나 활동적인지, 특이성, 그 활성이 다른 세포 처리에 민감한지 여부와 같은 키나아제 신호 전달 분야의 주요 질문에 답하는 데 도움이 될 수 있습니다. 이 기술의 주요 장점은 다재다능하고 정량적이라는 것입니다.

이 절차를 시연하는 사람은 이 분석에 대한 경험이 풍부한 Cobb Laboratory의 연구 과학자인 Steve Stippec입니다. 먼저 200마이크로리터의 세포 용해물에 1밀리리터당 1밀리그램 항체 2마이크로리터를 추가하고 흔들면서 섭씨 4도에서 1시간 동안 배양합니다. 용해 완충액을 추가해서 단백질 A-Sepharose 구슬을 2-3 번 세척하고 그 후에 5000 시간 g에 30 초에서 1 분 동안 4 °C에서 회전시키는 만지작거려, 그 후에 피펫으로 상등액을 제거하고 완충액에 있는 구슬을 다시 현탁하십시오.

View the full transcript and gain access to thousands of scientific videos