Affinity capture methods

Figure 7.4. Isolation of poly-A+ RNA by biotin-streptavidin affinity matrix. Poly-A+ RNA is captured as a hybrid between poly-A+ RNA and biotinylated oligo(dT) by streptavidin matrix. Most mRNAs carry poly-A+ stretch at their 3 end, and hence poly A-containing RNA can be enriched substantially by this affinity capture method. Poly-A+ RNA can be eluted from the beads by low salt or water. The eluted RNA can be ethanol precipitated.

Fig. 12.5. Triplex affinity capture method. The triplex is formed in slightly acidic conditions (I). The complex is captured on paramagnetic beads and washed (11) and the duplex eluted with a mild alkaline buffer.

The techniques developed to study protein interactions can be divided into a number of major categories (Table 31.1), including bioconjugation, protein interaction mapping, affinity capture, two-hybrid techniques, protein probing, and instrumental analysis (i.e., NMR, crystallography, mass spectrometry, and surface plasmon resonance). Many of these methods are dependent on the use of an initial bioconjugation step to discern key information on protein interaction partners. 

Figure 10.1 Principle of covalent capture methods. Drug fragments typically have weak binding affinity and can therefore be difficult to detect. By introducing two reactive groups, X and Y, a fragment that binds in the vicinity of X can be captured covalently by the protein target and easily identified by mass spectrometry.

In addition to finding small organic molecules that bind to a protein, covalent capture methods can identify peptides that interact with proteins. Kohda and colleagues used this approach to study the mitochondrial protein Tom20, an import receptor that recognizes an epitope on proteins targeted for the mitochondria.1301 Previous work had characterized this epitope as a five-residue peptide that assumes an amphiphilic helical conformation, and coarse sequence preferences had been worked out. However, Tom20 has both low affinity... 

Finally, using PNA as an affinity capture reagent recently was extended to probing RNA-protein complexes (RNPs) in cells (33). In this application, the PNA is functionalized with a peptide that allows uptake into cells and is complementary to an RNA component of an RNP. The PNA also bears two affinity tags, the first of which is a benzophenone-modifled phenylalanine residue that can photocross-link the PNA to a protein present in the RNP. The second tag is a biotin group, which allows the purification of the cross-linked PNA-protein. Subsequent analysis by mass spectrometry identifies both the protein and its cross-linking site. As is the case for PNA used to deliver a fluorophore to a specific site in an RNA, this method requires that the PNA not disrupt the structure being probed. 

Rheumatoid factors (Section 6.1.6.3) or an excess of IgG may interfere with this reaction. Moreover, IgM often have lower affinity for the antigen. An improvement of this method (Yolken et al., 1980), in which excess of IgG is removed from the test serum with SpA, is still not satisfactory. The elegant class capture methods can be illustrated for IgM as ... 

Li, F, Bamathan, E. S., and Karik6, K. (1994) Rapid method for screening and cloning cDNAs generated in differential mRNA display application of Northern blot for affinity capturing of cDNAs. Nucleic Acids Res. 22,1764,1765. 

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