Isolation of Whole Cell Protein Lysates from Mouse Facial Processes and Cultured Palatal Mesenchyme Cells for Phosphoprotein Analysis

Madison A. Rogers; Brenna J. C. Dennison; Katherine A. Fantauzzo

doi:10.3791/63834

Aislamiento de lisados de proteínas de células enteras de procesos faciales de ratón y células me...

JoVE Journal
Developmental Biology

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

JoVE Journal Developmental Biology

Isolation of Whole Cell Protein Lysates from Mouse Facial Processes and Cultured Palatal Mesenchyme Cells for Phosphoprotein Analysis

Aislamiento de lisados de proteínas de células enteras de procesos faciales de ratón y células mesenquimas palatinas cultivadas para el análisis de fosfoproteínas

Full Text

2,462 Views

07:26 min

April 1, 2022

DOI: 10.3791/63834-v

Madison A. Rogers*¹, Brenna J. C. Dennison*¹, Katherine A. Fantauzzo¹

¹Department of Craniofacial Biology, School of Dental Medicine,University of Colorado Anschutz Medical Campus

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

Overview

This protocol outlines a method for isolating whole cell protein lysates from mouse embryo facial processes and cultured mouse embryonic palatal mesenchyme cells. It facilitates the assessment of phosphorylated protein levels, crucial for understanding craniofacial development.

Key Study Components

Area of Science

Neuroscience
Developmental Biology
Cell Biology

Background

Phosphoproteins play vital roles in cellular signaling during development.
Protein isolation often leads to dephosphorylation, complicating analysis.
This protocol addresses these challenges in a relevant biological context.
Mouse models are commonly used to study craniofacial development.

Purpose of Study

To isolate proteins while preserving their phosphorylated states.
To analyze the dynamics of phosphoproteins in craniofacial development.
To provide a reliable method for studying embryonic protein functions.

Methods Used

Dissection of mouse embryos to obtain facial processes.
Culture of mouse embryonic palatal mesenchyme cells.
Use of phosphatase inhibitors during protein lysis.
Western blotting to assess phosphorylated protein levels.

Main Results

Successful isolation of proteins with preserved phosphorylation.
Insights into the role of phosphoproteins in craniofacial development.
Demonstration of the effectiveness of the protocol in relevant contexts.
Potential applications in further developmental biology research.

Conclusions

The protocol provides a robust method for studying phosphoproteins.
It enhances understanding of molecular mechanisms in craniofacial development.
Future studies can build on this method to explore related biological questions.

Frequently Asked Questions

What is the significance of phosphoproteins in development?

Phosphoproteins are crucial for signaling pathways that regulate cellular processes during development.

How does this protocol prevent dephosphorylation?

The use of phosphatase inhibitors in lysis buffers helps maintain the integrity of phosphorylated proteins.

What tissues can be analyzed using this method?

This method can be used for mouse embryo facial processes and cultured palatal mesenchyme cells.

What is the role of western blotting in this protocol?

Western blotting is used to assess the levels of phosphorylated proteins after isolation.

Can this protocol be adapted for other tissues?

While designed for craniofacial tissues, adaptations may be possible for other relevant tissues.

El protocolo presenta un método para aislar lisados de proteínas de células enteras de procesos faciales de embriones de ratón disecados o células mesenquimas palatales embrionarias de ratón cultivadas y realizar posterior western blotting para evaluar los niveles de proteína fosforilada.

Este protocolo se puede utilizar para obtener una mayor comprensión de la dinámica y el papel de las fosfoproteínas activas durante el desarrollo craneofacial de los mamíferos. Este protocolo supera la barrera común de la desfosforilación durante el aislamiento de proteínas de dos contextos fisiológicamente relevantes, los procesos faciales de ratón y las células de mesénquima palatal embrionarias de ratón cultivadas. Es imperativo moverse rápidamente mientras se mantienen todos los reactivos y materiales en hielo y usar inhibidores de la fosfatasa en todos los tampones de lisis para mantener la integridad de las proteínas fosforiladas.

Para comenzar, coloque el cuerpo del ratón en una tabla de disección con el lado ventral hacia arriba y rocíe el abdomen del ratón con etanol al 70%. Luego abra la cavidad abdominal pellizcando y levantando la piel anterior a la abertura vaginal con pinzas Semken rectas. A continuación, corte la piel levantada en las capas subyacentes con tijeras quirúrgicas de hoja recta en un ángulo de 45 grados a cada lado para generar una forma de V que se extienda a cada superficie lateral aproximadamente a medio camino entre las cuatro extremidades y las extremidades posteriores.

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

Explore More Videos

Biología del desarrollo Número 182 Desarrollo craneofacial fosforilación fosfoproteínas ratón procesos maxilares células mesenquimas palatinas embrionarias de ratón