Immunoprecipitation

During immunoprecipitation (IP), a protein of interest is pulled down with a specific antibody or Nanobody. The antibody is coupled to a matrix such as agarose or magnetic agarose and binds specifically to the protein of interest. Unbound sample content like proteins and lipids is washed away, while the precipitated protein is enriched on the matrix.

Immunoprecipitation is also a common and powerful technique for isolating a specific protein and its associated factors from cell or organelle extracts (see Co-Immunoprecipitation below).

ChIP, RIP, CLIP

ChIP, RIP, and CLIP are techniques for immunoprecipitation of nucleic acids via directly or indirectly interacting proteins.

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Co-Immunoprecipitation

Co-immunoprecipitation (Co-IP) is a method to identify the interacting partners (e.g. co-factors, ligands, inhibitors etc.) of a specific protein. Given that their interaction with the protein of interest is not too transient, the interaction partners are co-precipitated together with the protein that is pulled-down by the affinity resin.

Co-IP is a versatile technique for analyzing protein-protein interactions:

  • Identification of new interaction partners
  • Confirmation of interactions under various conditions

Nano-Traps are extraordinarily efficient tools for Co-IPs. In contrast to other affinity resins, the affinity ligand (i.e. the Nanobody) is covalently linked to the matrix, and virtually no protein comes off the Nano-Traps. Protein contaminants from the beads are frequently seen in IgG-based or streptavidin-based pull-downs and can mask potential interaction partners of the precipitated protein during SDS-PAGE or other methods of analysis. In addition, the small size and surface of the Nanobody reduces unspecific binding/background. Even during the proteolytic on-bead digest for MS sample preparation (see “Mass spectrometry ”), only few Nanobody peptides are released from the resin.

 

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Immunoprecipitation of Spot-tagged proteins

Spot-tagged proteins can be immunoprecipitated using Spot-Trap. The Spot-Tag can be placed either at the N-terminus or at the C-terminus of the protein of interest. Spot-Trap is available both as agarose beads or as magnetic agarose beads and is characterized by a remarkably low unspecific background binding.

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On-bead enzyme assays

In many cases, the enzymatic activity of an immunoprecipitated protein may be directly measured on the beads. It is recommended to precipitate the enzyme via a protein- or peptide-tag for the enzymatic assay to reduce steric hindrance.

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Mass spectrometry

When isolated by Co-IP, the interacting partners of a protein can be identified by MS; this is also called bottom-up proteomics. For MS sample preparation, these protein samples are denatured, reduced, alkylated, and digested. Then, resulting peptides are cleaned-up for analysis by MS, e.g. by peptide mass fingerprinting.

Nano-Traps are beneficial for MS analysis following Co-IP, because of their reliable performance, high affinity, and low background of protein contaminations. The superior stability of the Nano-Traps allows to apply stringent washing conditions. In most cases, even buffers containing chaotropic reagents or detergents can be used, whereas these would inactivate other IgG-based or Streptavidin-based affinity resins. Therefore, background can be further reduced, which increases the sensitivity of the sample analysis. This is required for applications like the analysis of some post-translational modifications (PTMs).

It is recommended to conduct the MS sample preparation on-bead to avoid loss of sample by incomplete elution from the Nano-Trap.

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Affinity purification

Next to small-scale immunoprecipitation, resin-coupled nanobodies can also be used for the purification of tagged recombinant proteins.

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Ubiquitination

Ubiquitination, also known as ubiquitylation, is a post-translational modification of a protein by adding ubiquitin molecules to the protein sequence. This enzymatic process is controlled by multiple enzymes. The 8.5 kDa ubiquitin is highly conserved among species. When conjugated to proteins, it tags those for degradation by the proteasome.

Reducing Background in ubiquitination assays

During ubiquitination assays when immunoprecipitating an ubiquitinated protein of interest, high background due to unspecific binding is frequently an issue. The considerable robustness of the GFP-Trap® allows to apply very harsh washing buffers and conditions to reduce background.

 

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Looking for references? Please check our literature database.

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