No/Weak Target Protein Precipitation

1. Weak/transient protein-protein interaction (no timely fixation);

2. Improper lysis buffer composition (excessive/insufficient detergent);

3. Over-washing leading to complex dissociation;

4. Extremely low protein expression

5. Excessively low antibody concentration;

1. Add cross-linker (e.g., DSP) to stabilize interactions, operate on ice throughout;

2. Adjust lysis buffer detergent concentration (0.1%–1% Triton X-100), add protease/phosphatase inhibitors;

3. Reduce washing cycles (3–4 times), lower wash buffer salt concentration;

4. Enrich samples or use high-sensitivity detection methods

5. Optimize the antibody dilution ratio (1:50 to 1:200);

High Non-Specific Background

1. Inadequate blocking;

2. Incomplete washing;

3. Protein sample degradation;

4. Non-specific adsorption by beads

5. Non-specific antibody binding;

1. Block beads with 5% BSA or non-fat milk for 1–2h;

2. Increase washing cycles (4–5 times), use wash buffer containing 0.1% Tween-20;

3. Operate on ice, add fresh inhibitors;

4. Pre-incubate beads with irrelevant protein (e.g., BSA) to reduce non-specific adsorption

5. set IgG isotype control;

Co-Immunoprecipitated Protein Not Detected

1. Unmet conditions for protein interaction (e.g., cofactor required);

2. Lysis buffer disrupts protein complex;

3. Insufficient detection antibody sensitivity;

4. Improper incubation time/temperature

1. Mimic physiological conditions (add necessary cofactors, adjust pH);

2. Reduce lysis buffer detergent strength, use mild lysis buffer (e.g., NP-40 lysis buffer);

3. Amplify the detection signall;

4. Incubate at 4℃ overnight to ensure sufficient interaction

Beads Aggregation/Incomplete Precipitation

1. Excessive beads concentration;

2. Insufficient binding between antibody and beads;

3. Inadequate shaking during incubation;

4. Insufficient centrifugation speed/time

1. Adjust beads volume (20–50μL per sample);

2. Incubate antibody with beads at room temperature for 1–2h before adding sample;

3. Incubate on shaker at low speed (50–100rpm);

4. Increase centrifugation speed (10000–12000rpm) or extend time (5–10min)

Protein Degradation

1. No/inactive inhibitors in lysis buffer;

2. Prolonged sample processing time;

3. Excessively high operating temperature;

4. Repeated freeze-thaw cycles of samples

1. Freshly add protease/phosphatase inhibitor cocktail;

2. Shorten sample processing time (<2h);

3. Operate on ice or at 4℃ throughout;

 4. Aliquot and store samples to avoid repeated freeze-thaw

False Positive Results (Erroneous Co-IP)

1. Antibody cross-reactivity;

2. Strong non-specific adsorption by beads;

3. Presence of protein aggregates in samples;

4. Too low salt concentration in wash buffer

1. Verify antibody specificity, use cross-adsorbed antibodies;

2. Optimize blocking and pre-incubation steps;

3. Add reducing agent (e.g., DTT) to lysis buffer to avoid protein aggregation;

4. Appropriately increase wash buffer salt concentration (150–300mM NaCl)