📋 Workflow
Full workflow, products add-to-cart directly1
Cell lysis
30 minLyse cells with gentle detergent lysis buffer to preserve protein interactions
💡 Tip Mild detergents preserve interactions
⚠️ Caution Keep cold with protease inhibitors
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2
Antibody coupling
2 hCouple specific antibody to Protein A/G beads or agarose, block non-specific binding
💡 Tip Include isotype IgG control
⚠️ Caution Rotate gently to avoid settling
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3
Co-immunoprecipitation
OvernightIncubate lysate with antibody-beads overnight to pull down target and its partners
💡 Tip Longer incubation enhances capture
⚠️ Caution Avoid harsh salt/detergent breaking interactions
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4
Wash, elute and analyze
2 hWash repeatedly to remove non-specifics, elute with elution buffer, analyze by SDS-PAGE/WB or MS
💡 Tip Verify interaction by WB with other antibodies
⚠️ Caution Elute thoroughly to avoid carryover
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⚠️ 5 Common Beginner Mistakes
- ① Non-specific binding causes high background
- ② Poor antibody specificity yields false interactions
- ③ Harsh detergent disrupts weak interactions
- ④ Insufficient washing leaves contaminants
- ⑤ Missing isotype control hinders specificity
❓ FAQ
+IP vs Co-IP?
IP enriches one target; Co-IP captures its partners to validate interactions.
+How to reduce background?
Pre-clear lysate, stringent washes, isotype control and optimal antibody.
+Magnetic beads or agarose?
Magnetic beads are faster/easier to separate; agarose suits classic protocols.