• Overview
• Affinity Isolation
• Affinity Purification
• Data Mining »
  • Vizualisations
  • Bioinformatics
  • Bead Proteomes & the PFL

a) General method

The affinity-purification of protein complexes relies upon the specific immuno-precipitation of a protein of interest (bait), along with its interaction partners, using antibodies, either to the endogenous bait protein, or to a tagged version of the bait, exogenously expressed in the cell. Antibodies are linked to bead matrices, including the commonly used Agarose, Sepharose and Magnetic beads.

The combination of affinity-purification techniques with MS has been widely developed as a highly sensitive, and unbiased, method to identify protein interaction partners and characterize the dynamics of protein complexes under different conditions (reviewed in (Gingras, Gstaiger et al. 2007; Collins and Choudhary 2008; Oeljeklaus, Meyer et al. 2009). The high sensitivity of MS technology increases the total number of proteins identified in each pull-down experiment. However, the majority of these proteins usually represent contaminants, e.g. proteins that bind non-specifically to the affinity matrix and/or to the antibody. In some cases, contaminants can represent more than 80% of the total number of proteins identified.

Therefore a major challenge of affinity-purification experiments relies upon the reliable discrimination between genuine protein interaction partners and non-specific contaminants. This is facilitated by:

• The optimization of the procedure to increase the IP efficiency (high specific signal).
• The reduction of the background of contaminants (low non-specific noise), and/or the “identification” of the contaminants.

b) Optimization of the IP protocol

Different approaches can be followed, which are described and compared below. They include the choice of the tag/antibody, the conjugation of antibodies to affinity matrices, the immuno-precipitation of specific protein complexes.

Choice of the tag/antibody
Both tag-based and endogenous pull-down experiments have advantages and disadvantages. Tagged baits provide a scalable and general method to identify specific protein interaction partners. Different types of tags are commonly used in affinity-purification experiments, such as fluorescent tags (e.g., GFP) and Flag tag. In particular, the GFP tag can be used in a dual strategy combining both fluorescence microscopy and affinity-purification (Trinkle-Mulcahy, Andersen et al. 2006). GFP has also proven to be an effective tag for affinity purification procedures, due 1) to its low background of non-specific interactions and 2) to the efficient recovery possible using recently developed “GFP-TRAP” (Chromotek) affinity matrices (Rothbauer, Zolghadr et al. 2008). All tags, however, can potentially affect protein structure, resulting in alteration of both protein function and association with binding partners. Counter this problem by the location of the tag, i.e., C and N terminal. In contrast, co-IP of endogenous proteins avoids several problems associated with the use of tags. However, this strategy relies on the availability of a specific and high affinity antibody that isolates the endogenous bait protein efficiently, which is often not available. Antibody affinity and specificity should always be checked carefully.

Conjugation of antibodies to affinity matrices
Antibodies are conjugated, covalently or not, to bead matrices (e.g., sepharose, agarose and magnetic beads). When combined to MS, it is highly recommended to covalently conjugate the antibody to the beads. Otherwise, a great amount of antibody can be eluted from the beads along with the specific protein complexes. Given that antibodies are usually in excess, as compared to cellular baits, they might strongly compete with other proteins for further MS identification.

The type of beads used for each pull-down experiment is an issue that is worth considering as the efficiency and cleanliness of different types of beads may vary according to the cell type and the type of extract used. In our experience, Dynabeads (Invitrogen) work well for nuclear extracts, whereas Sepharose and Agarose beads (GE-Healthcare) can give lower backgrounds when used with cytoplasmic extracts and whole cell extracts (Trinkle-Mulcahy, Boulon et al. 2008).

Immunoprecipitation protocol
To reduce the amount of non-specific binding proteins in a co-IP experiment, several options can be considered:

• a pre-clearing step (incubation of the cellular extract with bead matrices alone)
• incubation times kept to their minimum (1h max)
• high stringency buffer.

However, in our experience, increasing the buffer stringency can decrease the yield of protein recovered and risks losing low abundance and/or low affinity specific protein interaction partners, which are certainly the most difficult, but also the most interesting, proteins to identify.

Therefore, to preserve all genuine protein interaction partners, both stable and transient, we use medium or low stringency buffers.

As a result, many contaminants remain in the analysis, which need to be reliably identified and distinguished from the specific interaction partners.

Different methods have been developed to address this particular issue, both on the experimental and analysis levels.