mNeonGreen-Catcher

This protocol provides a general outline of how to use mNeonGreen-Catcher (agarose beads) for immunoprecipitation using a microcentrifuge for sedimentation. Alternatively, it is possible to use mNeonGreen-Catcher agarose beads in spin columns. All protocol steps should be carried out at 4 °C.

Protocol as PDF

1. For mammalian cells, harvest 10⁶-10⁸ cells per sample.
2. Lyse cells according to established protocols in 0.2 to 1.5 mL volume. Recommended Buffer Conditions: mNeonGreen-Catcher resins are compatible with commonly used Lysis and Washing buffers, e.g. RIPA buffer. The following buffer conditions have been tested:
   
   • pH ranging from pH 5 to pH 9
   • 2 % Triton X-100, 1 % Tween-20, 1 % NP-40, 1 % CHAPS, 1 % Deoxycholate, 0.1 % SDS
   • 4 M NaCl, 2 M KCl, 1 M MgCl₂
   • 100 mM EDTA
   • 4 M urea
   • 10 mM DTT, 10 mM 2-Mercaptoethanol
   • Protease Inhibitors
   • RNAse A, DNAse I, Benzonase
3. Centrifuge cell lysates in microcentrifuge tubes for 10 min at 14.000 x g at 4 °C. Keep a small samples as “input” fraction.
4. Transfer the supernatant to a fresh microcentrifuge tube for each sample and keep at 4 °C.
5. Homogenize the mNeonGreen-Catcher (agarose beads) slurry gently by shaking.
6. Transfer 20 μL bead slurry to a 1.5 mL microcentrifuge tube for each sample.
7. Add 1 mL Lysis Buffer to equilibrate mNeonGreen-Catcher (agarose beads).
8. Centrifuge mNeonGreen-Catcher (agarose beads) for 1 min at 1000 x g and carefully remove the supernatant.
9. Repeat wash steps once for a total of two washes.
10. Resuspend equilibrated mNeonGreen-Catcher (agarose beads) gently with the cell lysate supernatant.
11. Rotate the microcentrifuge tubes for 1 h at 4 °C.
12. Centrifuge microcentrifuge tubes for 1 min at 1000 x g at 4 °C. Keep a small sample as “unbound” fraction. Carefully remove the supernatant.
13. Resuspend mNeonGreen-Catcher (agarose beads) in 1 mL Lysis Buffer.
14. Centrifuge mNeonGreen-Catcher (agarose beads) for 1 min at 1000 x g and carefully remove the supernatant.
15. Repeat wash steps twice for a total of three washes.
16. Resuspend mNeonGreen-Catcher (agarose beads) gently in 1 mL TBS.
17. Centrifuge mNeonGreen-Catcher (agarose beads) for 1 min at 1000 x g and carefully remove the supernatant.
18. Resuspend mNeonGreen-Catcher (agarose beads) gently in 1 mL TBS.
19. Centrifuge mNeonGreen-Catcher (agarose beads) for 1 min at 3000 x g and carefully remove the supernatant.
20. Resuspend mNeonGreen-Catcher (agarose beads) resin in 50 µL 2X SDS samples buffer.
21. Heat mNeonGreen-Catcher (agarose beads) resin for 5 min to 95 °C.
22. Centrifuge microcentrifuge tubes for 1 min at 3000 x g and transfer the supernatant to fresh microcentrifuge tubes. Keep the mNeonGreen-Catcher (agarose beads) as backup.