Protein-Binding Arrays

Fig. 10. Quantification of ligand binding to arrayed proteins. Binding curves generated by measuring the relative amount of bound GADD45 DNA at increasing DNA concentrations allow binding affinities and specific activities to be calculated for each p53 variant in the array.

In a real biological system, DNA is mostly surrounded by many proteins. Protein binding to DNA involves a number of hydrogen bonds and electrostatic contacts between two biopolymers, and induces not only structural deviation from the typical B-form structure, but also electronic perturbation of the -stacked array of base pairs. We tackled the electronic effects of protein binding on the efficiency of hole transport by using a restriction en-... 

Shumaker-Parry JS, Aebersold R, Campbell CT (2004) Parallel, quantitative measurement of protein binding to a 120-element double-stranded DNA array in real time using surface plasmon resonance microscopy. Anal Chem 76 2071-2082... 

Without highly specific binding to proteins on the chip, thorough washing of the array may remove specific binders as well as non-specific binders. Protein bindings cannot offer a universal detection scheme like hybridization used in DNA arrays, which makes it difficult to discover enough probes for proteomics. 

The manufacture and processing of the protein microarray should be conducted in such a manner that the arrayed proteins remain in their native and active state. For most proteins, this usually means the hydrated state in order to avoid surface denaturation. For antibody arrays which are perhaps more forgiving than other proteins, it has been our experience that while these could be stored cold and dry, it is most important to rehydrate them prior to use. This process is in sharp contrast to the preparation of nucleic acid arrays in which strand melting or denaturahon is necessary to achieve optimal binding to the solid support. While the hybridization process is well understood and can be controlled under thermodynamic principles, the folding and renaturation of proteins on planar (microarray) surfaces is under study. 

The apparent permeability of 11 test compounds was measured in the presence and absence of human serum albumin in the donor compartment, and by solving the differential equations describing the kinetics of membrane permeability, membrane retention and protein binding, the authors were able to obtain the Kd. With the protein in solution rather than immobilized and without the need for mass balance or equilibrium conditions, this approach provides an attractive alternative to existing methods with the potential to be applied to an array of other soluble proteins. 

Tandem array of Binding sites for DnaA protein,... 

Most functions of DNA and RNA are dependent on proteins. Cells contain a vast array of polypeptides that bind to nucleic acids in many specific ways.387 390 We have seen (Fig. 5-21) that the histones hold super-coiled DNA in the form of nucleosomes391 in eukaryotic nuclei and similar proteins bind to and protect the... 

Fig. 6. Measurement of the relative amount of ligand bound to each protein in the array. (A) Schematic of on-chip binding assay in which a fluorescently labeled interaction partner binds to the functional, arrayed protein immobilized to the streptavidin-coated surface via the biotinylated BCCP tag. (B) p53 protein function microarray probed with Cy3-labeled GADD45 duplex oligo. Quantification of the signal intensity from each spot allows the effect of polymorphic and functional variation on the DNA binding function of p53 to be determined.

Houseman, B. T. and Mrksich, M. (2002) Carbohydrate arrays for the evaluation of protein binding and enzymatic modification. Chem. Biol. 9(4), 443-454. 

Protein Binding to the Lipid-Coated H50 ProteinChip Arrays... 

Fold Coat protein pVIII, single a helix packed in a helical array ssDNA-binding protein, antiparallel f3 sheet... 

Data on protein binding to each glycan in the array with links to their stmctures in CFG glycan stmctures DB. 

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