Imaging Polysomes Isolated from a Mouse Brain Using Atomic Force Microscopy

Abstract

Source: Lunelli, L., et. al. Peering at Brain Polysomes with Atomic Force Microscopy. J. Vis. Exp.(2016).

This video demonstrates the imaging of polysomes using atomic force microscopy. A nickel-ions-coated mica sheet anchors RNA with attached ribosomes. The sample is washed, dried, and mounted onto the microscope stage. The cantilever tip scans the sample surface, recording laser deflections to map the topology. Software is used to correct distortions and visualize high-resolution polysome structures.

Protocol

Caution: To avoid any RNA degradation of the samples, prepare all buffers using DEPC-treated water to minimize RNase contamination.

Results

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Cycloheximide	Sigma	1810	Prepararation of lysate
DNAseI	Thermo Scientific	89836	Prepararation of lysate
RiboLock RNAse Inhibitor	Life technologies	EO-0381	Prepararation of lysate
DEPC	Sigma	40718	Prepararation of lysate
Triton X100	Sigma	T8532	Prepararation of lysate
DTT	Sigma	43815	Prepararation of lysate
Sodium Deoxycholate	Sigma	D6750	Prepararation of lysate
Microcentrifuge	Eppendorf	5417R	Prepararation of lysate
Sucrose	Sigma	S5016	Sucrose gradient preparation
Ultracentrifuge	Beckman Coulter	Optima LE-80K	Sucrose gradient centrifugation
Ultracentrifuge Rotor	Beckman Coulter	SW 41 Ti	Sucrose gradient centrifugation
Polyallomer tube	Beckman Coulter	331372	Sucrose gradient centrifugation
Density Gradient Fractionation System	Teledyne Isco	67-9000-176	Sucrose gradient fractionation
AFM	Asylum Research	Cypher	Polysome visualization