docs/protocols/gentle_coip_v1.yaml

---
# Protocol metadata
id: PROT-0012
name: Gentle Co-Immunoprecipitation Protocol
version: 1.0
description: A gentle co-immunoprecipitation protocol for preserving protein-protein interactions
author: JM Jordan
created: 2023-01-01
last_updated: 2025-05-07
category: protein-analysis

# Materials required
materials:
  - name: Protein lysate
    amount: 3600 μg per condition
    preparation: Fresh or previously frozen
  - name: RIPA buffer
    preparation: With protease inhibitor tablets added fresh
    storage: 4°C
  - name: Antibody for immunoprecipitation
    amount: 2 μg per reaction
    storage: -20°C
  - name: Protein A/G beads
    preparation: 50% slurry in storage buffer
    storage: 4°C
  - name: PBS with 0.02% Tween
    preparation: Filter-sterilized
    storage: Room temperature
  - name: Non-denaturing loading buffer
    concentration: 1X
    storage: -20°C
  - name: LoBind tubes
    type: Protein low-binding microcentrifuge tubes
  - name: Liquid nitrogen
    amount: Sufficient for snap-freezing samples

# Equipment required
equipment:
  - name: Rotating mixer
    temperature: 4°C and room temperature capable
  - name: Refrigerated centrifuge
    settings: Low speed (1000g)
  - name: Magnetic rack
    type: For bead separation
  - name: 37°C heat block or water bath
    use: For elution step
  - name: -80°C freezer
    use: Sample storage

# Protocol steps
steps:
  - step: 1
    action: "Prepare protein lysate"
    details: "Prepare a lysate solution with 3600 μg of protein for each condition (e.g., HFA and HFB)"
  - step: 2
    action: "Adjust lysate volume"
    details: "Adjust each lysate to 1 mL total volume with RIPA buffer containing protease inhibitor tablets"
  - step: 3
    action: "Preclear lysate"
    details: "Add 50 μL of 50% bead slurry to each tube of lysate and rotate at 4°C for 1 hour"
  - step: 4
    action: "Remove beads"
    details: "Collect precleared lysate by centrifugation or magnetic separation"
  - step: 5
    action: "Aliquot lysate"
    details: "Divide each precleared lysate into 3 tubes (6 tubes total) and adjust each to 1 mL with RIPA buffer with inhibitors"
  - step: 6
    action: "Add antibody"
    details: "Add 2 μg of antibody to each tube"
  - step: 7
    action: "Incubate overnight"
    details: "Rotate tubes overnight at 4°C to allow antibody binding"
  - step: 8
    action: "Continue incubation"
    details: "Continue with room temperature rotation for 1 hour"
  - step: 9
    action: "Prepare beads"
    details: "Wash 600 μL beads with PBS + 0.02% Tween and resuspend in 600 μL RIPA buffer with inhibitors"
  - step: 10
    action: "Add beads to samples"
    details: "Add 100 μL of bead solution to each tube"
  - step: 11
    action: "Capture complexes"
    details: "Rotate at room temperature for 1 hour to capture antibody-protein complexes"
  - step: 12
    action: "Wash beads"
    details: "Wash beads 3X with 1 mL PBS + 0.02% Tween using gentle pipet mixing (no extended rotation)"
  - step: 13
    action: "Transfer beads"
    details: "After final wash, resuspend beads in 200 μL PBS + 0.02% Tween and transfer to fresh LoBind tubes"
  - step: 14
    action: "Elute proteins"
    details: "Add 30 μL 1X non-denaturing loading buffer and incubate at 37°C for 5 minutes"
  - step: 15
    action: "Collect eluate"
    details: "Separate solution from beads into new LoBind tubes"
  - step: 16
    action: "Freeze samples"
    details: "Snap freeze eluate in liquid nitrogen"
  - step: 17
    action: "Store samples"
    details: "Store at -80°C until analysis"

# Critical parameters
critical_parameters:
  - parameter: "Gentleness of handling"
    details: "Avoid harsh mixing or vortexing to preserve protein-protein interactions"
  - parameter: "Washing steps"
    details: "Brief, gentle washes without extended rotation periods minimize disruption of complexes"
  - parameter: "Temperature"
    details: "Maintain 4°C during initial binding to reduce non-specific interactions"
  - parameter: "Elution conditions"
    details: "Use non-denaturing conditions and mild temperature (37°C vs 95°C)"

# Controls
controls:
  - control: "IgG control"
    purpose: "Use matched isotype IgG instead of specific antibody to identify non-specific binding"
  - control: "Input sample"
    purpose: "Save an aliquot of pre-IP lysate to verify presence of proteins in starting material"
  - control: "Beads-only control"
    purpose: "Beads without antibody to identify proteins binding directly to beads"

# Troubleshooting
troubleshooting:
  - problem: "Low or no co-immunoprecipitated protein"
    solution: "Increase lysate amount; reduce washing stringency; verify antibody efficacy; use crosslinking"
  - problem: "High background"
    solution: "Increase preclearance time; use more stringent wash buffer; validate antibody specificity"
  - problem: "Degraded proteins"
    solution: "Add additional protease inhibitors; work more quickly; keep samples cold"

# Safety considerations
safety:
  ppe: "Lab coat, gloves, and eye protection required"
  hazards: "Liquid nitrogen causes freeze burns; handle with appropriate PPE and in well-ventilated area"

# Downstream applications
downstream_applications:
  - name: "Western blotting"
    preparation: "Add SDS sample buffer if needed; may need to optimize antibody to avoid IP antibody detection"
  - name: "Mass spectrometry"
    preparation: "Consider specific elution methods compatible with MS analysis"

# References
references:
  - "Lin YC, et al. (2018) Optimization of a Co-Immunoprecipitation Protocol for the Detection of Weak Protein-Protein Interactions. PLoS ONE 13(10):e0206167"
  - "Antrobus R & Borner GH. (2011) Improved elution conditions for native co-immunoprecipitation. PLoS ONE 6(3):e18218"

# Notes
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
  - Remember to freeze Input and leftover samples
  - For weaker interactions, consider chemical crosslinking before lysis
  - Non-denaturing loading buffer preserves complexes for native gel electrophoresis
  - Alternative elution methods include peptide competition or pH elution
  - For subsequent SDS-PAGE analysis, an aliquot can be mixed with standard denaturing loading buffer
---