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A polyhistidine-tag, best known by the trademarked name His-tag, is an amino acid motif in proteins that typically consists of at least six histidine (His) residues, often at the N- or C-terminus of the protein. It is also known as a hexa histidine-tag, 6xHis-tag, or His6 tag. The tag was invented by Roche, although the use of histidines and its vectors are distributed by Qiagen. Various purification kits for histidine-tagged proteins are commercially available from multiple companies.
Protein purification is a series of processes intended to isolate one or a few proteins from a complex mixture, usually cells, tissues or whole organisms. Protein purification is vital for the specification of the function, structure and interactions of the protein of interest. The purification process may separate the protein and non-protein parts of the mixture, and finally separate the desired protein from all other proteins. Ideally, to study a protein of interest, it must be separated from other components of the cell so that contaminants will not interfere in the examination of the protein of interest's structure and function. Separation of one protein from all others is typically the most laborious aspect of protein purification. Separation steps usually exploit differences in protein size, physico-chemical properties, binding affinity and biological activity. The pure result may be termed protein isolate.
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Surface-assisted laser desorption/ionization (SALDI) is a soft laser desorption technique used for mass spectrometry analysis of biomolecules, polymers, and small organic molecules. In its first embodiment Koichi Tanaka used a cobalt/glycerol liquid matrix and subsequent applications included a graphite/glycerol liquid matrix as well as a solid surface of porous silicon. The porous silicon represents the first matrix-free SALDI surface analysis allowing for facile detection of intact molecular ions, these porous silicon surfaces also facilitated the analysis of small molecules at the yoctomole level. At present laser desorption/ionization methods using other inorganic matrices such as nanomaterials are often regarded as SALDI variants. As an example, silicon nanowires as well as Titania nanotube arrays (NTA) have been used as substrates to detect small molecules. SALDI is used to detect proteins and protein-protein complexes. A related method named "ambient SALDI" - which is a combination of conventional SALDI with ambient mass spectrometry incorporating the direct analysis real time (DART) ion source has also been demonstrated. SALDI is considered one of the most important techniques in MS and has many applications.
A peptide microarray is a collection of peptides displayed on a solid surface, usually a glass or plastic chip. Peptide chips are used by scientists in biology, medicine and pharmacology to study binding properties and functionality and kinetics of protein-protein interactions in general. In basic research, peptide microarrays are often used to profile an enzyme, to map an antibody epitope or to find key residues for protein binding. Practical applications are seromarker discovery, profiling of changing humoral immune responses of individual patients during disease progression, monitoring of therapeutic interventions, patient stratification and development of diagnostic tools and vaccines.
Affimer molecules are small proteins that bind to target proteins with affinity in the nanomolar range. These engineered non-antibody binding proteins are designed to mimic the molecular recognition characteristics of monoclonal antibodies in different applications. These affinity reagents have been optimized to increase their stability, make them tolerant to a range of temperatures and pH, reduce their size, and to increase their expression in E.coli and mammalian cells.
In the medical field of immunology, nanoCLAMP affinity reagents are recombinant 15 kD antibody mimetic proteins selected for tight, selective and gently reversible binding to target molecules. The nanoCLAMP scaffold is based on an IgG-like, thermostable carbohydrate binding module family 32 (CBM32) from a Clostridium perfringens hyaluronidase. The shape of nanoCLAMPs approximates a cylinder of approximately 4 nm in length and 2.5 nm in diameter, roughly the same size as a nanobody. nanoCLAMPs to specific targets are generated by varying the amino acid sequences and sometimes the length of three solvent exposed, adjacent loops that connect the beta strands making up the beta-sandwich fold, conferring binding affinity and specificity for the target.
References
- ↑ Mikhailov, V. S.; et al. (January 2013). "Affinity capture of specific DNA fragments with short synthetic sequences". Russian Journal of Bioorganic Chemistry. 39 (1): 72–76. doi:10.1134/S106816201301007X. PMID 23844509. S2CID 254980916.
- ↑ Johnson, Erin M.; et al. (1 August 2009). "Affinity capture mass spectrometry of biomarker proteins using peptide ligands from biopanning". Analytical Chemistry. 81 (15): 5999–6005. doi:10.1021/ac900868q. PMC 2846195 . PMID 19572565.
- ↑ LaCava, J; et al. (1 July 2016). "Protein Complex Purification by Affinity Capture". Cold Spring Harbor Protocols. 2016 (7): pdb.top077545. doi:10.1101/pdb.top077545. PMID 27371601.
- ↑ Tran, John C.; et al. (2016). "Automated Affinity Capture and On-Tip Digestion to Accurately Quantitate in Vivo Deamidation of Therapeutic Antibodies". Analytical Chemistry. 88 (23): 11521–11526. doi:10.1021/acs.analchem.6b02766. PMID 27797494.
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