Immunoprecipitation (IP) protocol

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Immunoprecipitation (IP)



Immunoprecipitation technique is developed from the specific antigen-antibody interaction and specific binding of bacterial protein A/G to FC fragment of antibody (immunoglobulin). As most frequently performed, protein A/G is firstly binded to agarose beads, then reacts with mixture of sample solution (antigen) and antibody. Thus protein A/G on the beads adsorbs the target antigen which can be easily isolated from thousands of protein antigens in sample solution by low-speed centrifuge.


Co-Immunoprecipitation, Co-IP

Chromatin immunoprecipitation, ChIP

RNA immunoprecipitation, RIP






Nowadays some products change the agarose beads to magnetic beads. That facilitates washing and has low-background and need a elution step, but a good choice anyway. (e.g. Dynabeads by Invitrogen)

Due to a long series of procedure and a denaturing experimental condition, to obtain a perfect result you need not only high-quality antibodies but a strict quota control for the IP system is also required. In a typical IP process, from incubation of antibody and agarose beads, washing of antibody-agarose beads complex to the final identification, each step is pivotal.


Antigen & Antibody


Troubleshooting for IP



Reagents and buffers:

RIPA lysis buffer:

  • 10mM Tris-HCl pH=7.4
  • 150mM NaCl
  • 5mM EDTA pH=7.4
  • 1% TritonX-100
  • 1% DOC
  • 0.1% SDS
  • 0.1mM TLCK
  • 0.2mM TPCK

protein A/G-agarose beads, specific primary antibody

Standard protocol (4 steps):

1. Sample treatment:

Harvest your cells

Add in appropriate amount of lysis buffer for cell IP (containing proteinase inhibitor)

Allow 30 min for a complete lysis at 4°C or on ice

Centrifuge at 12,000 g for 30 min and keep the supernatant.

For more details of lysis buffer and tips, please click here

2. Incubation of antibody-beads:

Keep a little amount of lysate for a later western-blot analysis.

Add in 1μg specific antibody and 10-50 μl protein A/G-beads

Incubate on a low-speed horizontal shaker for overnight at 4°C

For more details and tips of this step, please click here

3. Washing of antibody-agarose beads complex:

After the immunoprecipitation reaction, centrifuge at 3,000g 4°C for 5 min, then protein A/G-beads complex should be at the bottom of each tube.

Carefully remove the supernatant and wash the protein A/G-beads complex with 1ml lysis buffer for 3-4 times

(if needed)Add in appropriate amount of SDS loading buffer and keep samples in boiling water for 10 min

For more details and tips of this step, please click here

4. Final identification:

Proceed to SDS-PAGE, western-blot or mass spectra analysis.

For more details and tips of this step, please click here