Biomolecules Oligonucleotides, Proteins

The constitution of repetitive biomolecules like proteins or oligonucleotides can be detected by more sensitive methods than NMR. Thus, NMR is not the way to determine the sequence of a protein or RNA. This is simply because NMR requires some hundred nanomoles of substance of repetitive biomolecules, and other methods such as Edman degradation, mass spectroscopy, or oligonucleotide sequencing are much more sensitive. 

In many affinity techniques, biomolecules such as DNA, oligonucleotides, proteins, peptides, cofactors, and other biochemical compounds are used as reagents. Mainly proteins and, in particular, antibodies are discussed herein, due to their broad applicability. However, many rules can be easily transferred to other binding molecules. 

Once a molecule is modified with a hydrazine reagent and another molecule is modified with the benzaldehyde compound, they may be combined to form the final conjugate, which will result in a hydrazone linkage between the two molecules. In addition, chemoselective ligation using aldehyde/hydrazine reactions may be done to immobilize biomolecules. In this regard, one modified component may be a surface and the other one an antibody, protein, or oligonucleotide destined for immobilization onto the surface. 

In order to facilitate analysis of FeBABE produced fragments, the prey protein or biomolecule is labeled at one end with a tag that can be detected after electrophoresis, usually in a transfer blot. The tag can be a fusion tag, such as 6X His, or any other group that can be targeted with an antibody and detected. Alternatively, radiolabels and fluorescent labels have been used with prey molecules, including the use of end-labeled DNA to study where DNA binding proteins dock onto the oligonucleotide sequence. 

Certain biomolecules can be added into the bead or be attached to its surface. These mostly include such recognition elements as antibodies, oligonucleotides or other receptors such as conconovalin A. Enzymes can be used to design biosensors (e.g., for glucose) on a microscale but this research is still in its infancy. Finally, fluorescent proteins can be used as alternative to the dyes. The same refers to quantum dots which can also be used in principle. 

The third group of Au NP-biomolecule interactions are referred to as specific affinity interactions and include nanoparticles functionalized with groups that provide affinity sites for the binding of bio molecules such as proteins and oligonucleotides. 

Capillary electrophoresis (CE) coupled to MS has the advantage of high resolution and soft ionization for biomolecules, which may be used to differentiate post-translational modifications and variants of intact proteins and oligonucleotides. Different modes of CE (capillary zone electrophoresis, capillary isoelectric focusing, capillary electrochromatography, micellar electrokinetic chromatography, nonaqueous capillary electrophoresis) to MS as well as online preconcentration techniques (transient capillary isotachophoresis, solid-phase extraction, membrane preconcentration) are used to compensate for the restricted detection sensitivity of the CE methodology [77, 78]. 

---