assay protocol

Immunoprecipitation (IP)

Principle:

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.

IP
Category:
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.

► Applications ► Antigen & Antibody
► FAQ ► 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