---
name: Gentle Co-Immunoprecipitation Protocol
id: PROT-0012
version: 1.0
description: A gentle co-immunoprecipitation protocol for preserving protein-protein interactions
author: JM Jordan
created: 2023-01-01
materials:
  - Protein lysate
  - RIPA buffer with protease inhibitor tablets
  - Antibody (2 µg per reaction)
  - Beads (50% slurry)
  - PBS with 0.02% Tween
  - Non-denaturing loading buffer (1X)
  - LoBind tubes
  - Liquid nitrogen
steps:
  - "Prepare protein lysate (3600 µg protein)"
  - "Preclear with beads"
  - "Add antibody and incubate overnight at 4°C"
  - "Add washed beads and incubate"
  - "Wash beads with PBS-Tween"
  - "Elute proteins with non-denaturing buffer"
  - "Store samples at -80°C"
notes: |
  This is a gentle protocol designed to preserve protein-protein interactions
  Uses minimal washing steps without extended rotations
  Includes preclearance step to reduce non-specific binding
---

#Protocol 
by JM Jordan 2023

1. Prepared a lysate solution with 3600 ug of protein for HFA and HFB
    
2. Adjusted to 1mL each with RIPA+Tablets
    
3. Precleared solution by adding 50ul of 50% beads to each tube of lysate
    
4. Aliquoted into 3 tubes/lysate (6 tubes total) and adjusted each to 1mL with RIPA+Tablets
    
5. Added 2 ug of antibody to each tube
    
6. Rotated overnight at 4C
    
7. RT rotation for 1h
    
8. Washed 600ul beads with PBS + 0.02% Tween
    
9. Resuspended beads in 600ul RIPA+Tabs
    
10. Added 100ul of bead solution to each tube
    
11. Rotated at RT for 1h
    
12. Washed beads 3X with 1ml PBS + 0.02% Tween with gentle pipet mixing (no 5 min rotation)
    
13. After final wash, resuspended beads in 200 ul PBS + 0.02% Tween and transferred to fresh LoBind tubes
    
14. Added 30 ul 1X Non denaturing loading buffer and eluted as usual at 37C for 5 min
    
15. Separated solution from beads into new LoBind tubes and snap froze in LN2 and stored at -80C.
    
16. Note: Freeze Input and leftover