# Protocol for identifying components of subcellular compartments by antibody-based in situ biotinylation

## TL;DR

This paper introduces a protocol using antibody-based biotinylation to identify components of membrane-less subcellular organelles for comprehensive analysis.

## Contribution

A novel protocol for in situ biotinylation to identify subcellular compartment components using HRP-conjugated antibodies.

## Key findings

- The protocol enables labeling and purification of biotinylated components from subcellular compartments.
- It supports high-throughput analysis of DNAs, RNAs, and proteins in nuclear bodies.
- The method is applicable for studying dynamic membrane-less organelles.

## Abstract

Recent studies revealed that membrane-less subcellular organelles play important roles in cellular functions. Here, we present a protocol for identifying subcellular compartment components by antibody-based in situ biotinylation. We describe steps for in situ biotinylation labeling using a horseradish peroxidase (HRP)-conjugated antibody, purification of the biotinylated components, and sample preparation for high-throughput analysis. This protocol has potential for application in the comprehensive analysis of dynamic subcellular organelles.

For complete details on the use and execution of this protocol, please refer to Noguchi et al.1

•Steps for in situ biotinylation to identify the components of nuclear bodies•Instructions on identifying DNAs, RNAs, and proteins in nuclear bodies•Procedures for preparing biotinylated proteins for MS or DNAs/RNAs for NGS

Steps for in situ biotinylation to identify the components of nuclear bodies

Instructions on identifying DNAs, RNAs, and proteins in nuclear bodies

Procedures for preparing biotinylated proteins for MS or DNAs/RNAs for NGS

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Recent studies revealed that membrane-less subcellular organelles play important roles in cellular functions. Here, we present a protocol for identifying subcellular compartment components by antibody-based in situ biotinylation. We describe steps for in situ biotinylation labeling using a horseradish peroxidase (HRP)-conjugated antibody, purification of the biotinylated components, and sample preparation for high-throughput analysis. This protocol has potential for application in the comprehensive analysis of dynamic subcellular organelles.