Aug 22, 2025

BEAD INJECTION PROTOCOL V.2

DOI
https://dx.doi.org/10.17504/protocols.io.5jyl88j46l2w/v2
  • Yuanzhuo Zhou1,2,
  • Kohei Asai1,2,
  • Hirohisa Kyogoku1,3,
  • Tomoya S. Kitajima1,2
  • 1Lab for Chromosome Segregation, RIKEN Center for Biosystems Dynamics Research (BDR), Kobe, Japan;
  • 2Graduate School of Biostudies, Kyoto University, Kyoto, Japan;
  • 3Graduate School of Agricultural Science, Kobe University, Kobe, Japan
  • Designing protein-based artificial kinetochore
Yuanzhuo Zhou
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DOI: https://dx.doi.org/10.17504/protocols.io.5jyl88j46l2w/v2
Protocol Citation: Yuanzhuo Zhou, Kohei Asai, Hirohisa Kyogoku, Tomoya S. Kitajima 2025. BEAD INJECTION PROTOCOL. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl88j46l2w/v2Version created by Yuanzhuo Zhou
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 21, 2025
Last Modified: August 22, 2025
Protocol  Integer ID: 225234
Keywords: bead injection protocol, bead injection protocol this protocol, bead injection, mouse oocyte, protocol
Abstract
This protocol describes workflow of the bead injection in mouse oocytes.
Materials
Rat anti-GFP antibody (D153-3, MBL), PBS, MonoMag Protein G-conjugated beads (1 µm diameter, MPG1000, or 3 µm diameter, MPG3000, Ocean NanoTech), mRNA-injected GV-stage oocytes, piezo pulse device.
Protocol
Prepare Rat anti-GFP antibody (D153-3, MBL) at 1:30 dilution in PBS.
Add MonoMag Protein G-conjugated beads (1 µm diameter, MPG1000, or 3 µm diameter, MPG3000, Ocean NanoTech) at 1:20 dilution to the antibody solution.
Incubate the mixture for 2.5 hours at 4 °C with rotation to immobilize the antibody to the beads.
Wash the antibody-bound beads with PBS.
Store the antibody-bound beads in PBS until use.
Introduce the anti-GFP-bound beads into mRNA-injected GV-stage oocytes by microinjection using a piezo pulse.
Inject either 4 beads (3 µm diameter) or 4–11 beads (1 µm diameter) per oocyte.
Culture the microinjected oocytes at 37 °C for 1 hour before meiotic resumption.