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Immunoprecipitation (IP)

A technique used to selectively isolate and concentrate a specific protein or group of proteins from a complex mixture, such as a cell lysate. The primary purpose of IP is to study protein-protein interactions, post-translational modifications, and protein functions. By pulling down a target protein along with its interacting partners, researchers can identify and characterize these interactions.

Workflows requiring Immunoprecipitation

 

Top Methods for  Immunoprecipitation

 

Classic IP (Immunoprecipitation)

Isolate a specific protein from a complex mixture, allowing for the study of its properties, post-translational modifications, or for further analysis.

Procedure:

  1. Cells are lysed using lysis buffers to release proteins.
  2. A specific antibody, tailored to the target protein, is attached to beads (either agarose or magnetic).
  3. The cell lysate is incubated with the antibody-bead complex, allowing the target protein to bind.
  4. Non-specifically bound proteins are washed away using wash buffers.
  5. The target protein is then eluted from the beads using elution buffers.
  6. Bead-bound proteins are separated from the lysate using a centrifuge or magnetic stand.

 

Co-Immunoprecipitation (Co-IP)

Study protein-protein interactions by isolating a primary protein and its potential interacting partners from a complex biological sample.

Procedure:

  1. Cells are lysed to release proteins.
  2. A specific antibody, tailored to the primary protein, is attached to beads.
  3. The cell lysate is incubated with the antibody-bead complex.
  4. Proteins interacting with the primary protein will co-precipitate during this step.
  5. Non-specifically bound proteins are washed away.
  6. The primary protein and its interactors are eluted.
  7. Bead-bound proteins are separated using a centrifuge or magnetic stand.

 

Tandem Affinity Purification (TAP)

Achieve high purity purification of protein complexes, especially useful for studying low-abundance or transient protein-protein interactions.

Procedure:

  1. A protein of interest is genetically modified to include a dual-tag system.
  2. Cells expressing this modified protein are lysed.
  3. The lysate is first incubated with beads that bind to the first tag, isolating the protein.
  4. The first tag is cleaved off using a specific protease.
  5. A second round of purification is done using beads that bind to the second tag.
  6. After this step, the protein complex is eluted with high purity.
  7. Bead-bound proteins are separated using a centrifuge.

Why is Immunoprecipitation difficult:

Immunoprecipitation has never been easier

The OT-2 is a bench-top liquid handler designed to be accessible and flexible enough to automate many common applications.

How to Automate the Immunoprecipitation process:

Benefits of Automation over Manual Pipetting for Immunoprecipitation:

Resource Spotlights

Opentrons helps you automate Immunoprecipitation with open-source protocols for the OT-2 and Opentrons Flex

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