NUCLEIC ACID PROGRAMMABLE PROTEIN ARRAY
Fabrication of protein arrays usually requires lengthy preparation times along with laborious separation procedures, although there are methods, which can speed up this process and some extent cope with this issue. As per the timeline development and manufacturing of protein arrays followed the development of DNA arrays and therefore the knowledge from DNA arrays was applied to protein array production.Distance 80µm
The major hassles that restrict protein detection represent alterations in protein structures, interactions, instability, or loss of protein functionality during the analysis. Manufacturing of protein arrays is accompanied by numerous challenges ranging from tedious purification steps, obtaining a dynamic range in yields, to the risk of protein unfolding or in the worst case, denaturation, and loss of functionality during manipulations.Distance 100µm
The core of the protein array is its immobilization to the surface. Proteins can be immobilized on a microarray chip by adopting various strategies like physical adsorption by a covalent or non-covalent bond. The hydrophilic layer applied on the surface of choice is usually functionalized with specific chemistries and affinity tags complimenting the functional groups linked to the biomolecule to be immobilized. Typically proteins are characterized by a hydrophobic core with hydrophilic amino acids generally projected on the surface. That’s these hydrophilic amino acids modified with appropriate affinity tags that interact with the complimenting functional groups on the surface of the chip and thereby establish protein immobilization. Depending on the type of protein, its confirmation, and physical-chemical properties the process of immobilization is tweaked and optimized, usually by adjusting pH or temperature of buffers. In order to avoid the necessity of protein purification, problems with protein stability during storage and to have sufficient amounts of functional protein for the study an alternate mode of fabricating protein microarrays was devised by printing complementary DNAs. The hallmark of this approach was that the target proteins got translated and the epitope tags fused to the proteins allowing them to be immobilized in situ.
A novel method of printing protein microarray was developed i.e. - the Nucleic Acid Programmable Protein Array (NAPPA). In this approach, the proteins are synthesized from a DNA template on the microarray substrate in situ where the DNA template has been spotted and the new protein is held by affinity reagents. The method is primarily based on using cDNA clones in expression plasmids. The biggest advantage is gaining gene integrity for an unlimited period, ensuring identity, as the clones are verified by sequencing. Next to this, there is a possibility to incorporate the tags by inserting a clone into a plasmid. Bacteria cultures serve as hosts for the production of plasmid DNA, and after purification, these recombinants are printed on the surface. Cell-free expression systems produce a protein without using living cells. This means that these new expression clones are spotted on the array and the proteins are produced in situ in a cell-free system and subsequently immobilized in place on completion of their synthesis. This method is characterized by minimal manipulations with proteins, reduced risks of protein instability, and avoiding protein purification procedures thereby saving time and enhancing throughput.
Contact spotting by pinspotter
Plasmids coding for the protein are bound to a polyclonal antibody, that is immobilized on the surface of the array and the final detection of the protein is done by the monoclonal antibody. Synthesis of the protein in situ prevents the sample from damage during handling. This method provides the versatility to print protein arrays on diverse surfaces, MTPs, beads, or membranes and also provides the flexibility to detect different protein moieties ranging from antibodies to cell lysates.Microarray reader
The results are detected robotically by fluorescence or chemiluminescence scanners. These arrays are usually used for the detection of higher protein numbers, usually several hundred or thousands, this is the reason why pin spotter would be the best option for this application.Microarray contact spotting by M2-Automation pinspotter
NAPPA is the only in situ protein technology, that has been used in biological and biomedical studies. It is getting an important method for screening biomarkers, due to its high sensitivity and specificity. This technology is becoming crucial for identifying and characterizing proteins that regulate events in the progression of diseases like diabetes type I, breast, and lung cancer. Presently, protein microarrays are used in the biomarker studies, post-translational protein modifications, and for studying different types of interactions with proteins.