Method For Immobilization Of Proteins Research Articles

Advances of Methods of Protein Immobilization on Capillary Column

Abstract In fused silica capillary, immobilized proteins (enzymes) have important applications in chiral separation, peptide mapping, drug screening, preconcentration and so on. In this article, we reviewed various methods for protein immobilization in capillary, including sol-gel method, physical adsorption, ion chelate adsorption, lipid-based protein immobilization and covalently bound.

A comparative investigation of methods for protein immobilization on self-assembled monolayers using glutaraldehyde, carbodiimide, and anhydride reagents

Three different approaches to the immobilization of proteins at surfaces have been compared. All rely on the creation of surface groups that bind primary amines on lysine residues. Carboxylic acid terminated self-assembled monolayers (SAMs) have been activated using a water soluble carbodiimide to yield an active ester functionalized surface and with trifluoroacetic anhydride to yield a surface anhydride, and amine terminated SAMs have been activated using glutaraldehyde. Although the degree of surface derivatization by n-alkylamines was greater using the carbodiimide and anhydride methods under anhydrous conditions, the glutaraldehyde activation of amine terminated SAMs yielded significantly greater attachment of streptavidin than is achieved using either of the other methods. This is attributed to the susceptibility to hydrolysis of the active species formed by activation of the carboxylic acid terminated monolayers. Patterned protein structures may be formed by using both glutaraldehyde activation of amine terminated thiols and carbodiimide activation of carboxylic acid terminated thiols, in conjunction with selective photo-oxidation of oligo(ethylene glycol) terminated SAMs.

Evaluation of Electrochemical-based Bio-lithography Method for Immobilization of Proteins on a QCM Array

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Enhanced bone morphogenetic protein‐2 performance on hydroxyapatite ceramic surfaces

The immobilization of biomolecules on biomaterial surfaces allows for the control of their localization and retention. In numerous studies, proteins have been simply adsorbed to enhance the biological performance of various materials in vivo. We investigated the potential of surface modification techniques on hydroxyapatite (HA) ceramic discs in an in vitro approach. A novel method for protein immobilization was evaluated using the aminobisphosphonates pamidronate and alendronate, which are strong Ca chelating agents, and was compared with the established silanization technique. Lysozyme and bone morphogenetic protein-2 (BMP-2) were used to assess the suitability of the two surface modification methods with regard to the enzymatic activity of lysozyme and to the capacity of BMP-2 to stimulate the osteoblastic differentiation of C2C12 mouse myoblasts. After immobilization, a 2.5-fold increase in enzymatic activity of lysozyme was observed compared with the control. The alkaline phosphatase activity per cell stimulated by immobilized BMP-2 was 2.5-fold higher [9 x 10(-6) I.U.] than the growth factor on unmodified surfaces [2-4 x 10(-6) I.U.]. With regard to the increase in protein activity, both procedures lead to equivalent results. Thus, the bisphosphonate-based surface modification represents a safe and easy alternative for the attachment of proteins to HA surfaces.

Preparation of Porous Protein‐Based Hydogel for Highly Sensitive Protein Chips

AbstractSummary:Protein chips are important tools for high‐throughput analysis of biological events. We have developed a novel method to prepare a protein‐based hydrogel, that is, a “Three‐Dimensional Nano‐structured Protein Hydrogel” (3‐D NPH), which is composed of protein and polymer nano‐particles. The 3‐D NPH could be easily prepared by dispensing a protein and polymer mixture on a substrate. Surprisingly, gold particles conjugated with protein A diffused into the 3‐D NPH which was made of mouse IgG through the pores. We have shown that the protein chips made with our 3‐D NPH method has tremendously improved sensitivity in detecting protein‐protein interactions compared with that of direct protein immobilization methods.

Solid-phase assays for small molecule screening using sol-gel entrapped proteinsThis paper is one of a selection of papers published in this Special Issue, entitled CSBMCB — Systems and Chemical Biology, and has undergone the Journal's usual peer review process.

With compound libraries exceeding one million compounds, the ability to quickly and effectively screen these compounds against relevant pharmaceutical targets has become crucial. Solid-phase assays present several advantages over solution-based methods. For example, a higher degree of miniaturization can be achieved, functional- and affinity-based studies are possible, and a variety of detection methods can be used. Unfortunately, most protein immobilization methods are either too harsh or require recombinant proteins and thus are not amenable to delicate proteins such as kinases and membrane-bound receptors. Sol-gel encapsulation of proteins in an inorganic silica matrix has emerged as a novel solid-phase assay platform. In this minireview, we discuss the development of sol-gel derived protein microarrays and sol-gel based monolithic bioaffinity columns for the high-throughput screening of small molecule libraries and mixtures.

Directed immobilization of DNA-binding proteins on a cognate DNA-modified chip surface

Here we describe a useful method for the site-directed immobilization of proteins with a DNA-binding domain (DNA-BD) on the cognate DNA-coated gold surface for surface plasmon resonance (SPR) imaging analyses. In order to assess the performance of this procedure, we utilized two DNA-BDs, yeast GAL4 DNA-BD, and bacterial LexA DNA-BD. After the immobilization of the cognate double-stranded DNAs (dsDNAs) to a gold chip surface with a monolayer of poly( l-lysine) for sequence-specific DNA–protein interaction, purified recombinant GAL4 DNA-BD:EGFP and LexA DNA-BD:RFP fusion proteins were applied to a dsDNA-spotted gold chip, and were subsequently analyzed using an SPR imaging system. Consequently, the recombinant DNA-binding proteins, GAL4 DNA-BD:EGFP and LexA DNA-BD:RFP, were shown to bind selectively to their cognate DNA sequences on the gold chip. Collectively, our results revealed that sequence-specific dsDNA microarray approach could prove useful in performing the site-directed immobilization of DNA-binding proteins onto a gold thin film in a parallel format, and thereby potentially allowing for the analysis of transcription factor binding profiling as well as for the monitoring of protein–protein interactions between target proteins with DNA-binding domain as a fusion tag and their binding partners.

Localized immobilization of proteins onto microstructures within a preassembled microfluidic device

We describe herein a method for the site-specific immobilization of proteins on a three-dimensional polydimethylsiloxane (PDMS) microstructure, i.e., an array of microposts, within the channel of a microfluidic device. The protein-adhesive sites in the preassembled device are protected by a layer of heparin, an antibiofouling agent. To ready a device for experimentation, electrical pulses from microelectrodes within the channel spatiotemporally generate hypobromous acid that quickly removes the heparin, exposing protein-adhesive surface. To prove that, for assays performed within microfluidic channels of identical dimensions, their relative sensitivities can be increased if the inner channel surface areas are increased by the presence of PDMS microstructures, a glutathione peroxidase (GPX) sandwich immunoassay was performed within a microfluidic channel that had a region without and a region with a microstructure. Even though both regions had the same two-dimensional areas, the added dimension of the PDMS microstructure significantly increased the sensitivity of the GPX assay. Finally, irregularly shaped, protein-adsorptive regions occur upon electrochemical treatment when there is fluid-flow even in the absence of moving liquid. We found that the shape of protein-adsorptive regions can be completely controlled, even in the presence of fluid-flow, when the protein-adsorptive regions are delineated by regions of poly(ethylene glycol)dimethacrylate.

The response of osteoblast‐like cells towards collagen type I coating immobilized by p‐nitrophenylchloroformate to titanium

The scaffold surface composition can be altered by the use of surface coatings. The use of thin coatings will give special surface properties, while the bulk properties of the scaffold are preserved. Collagen type I is known to play an important role during cell adhesion as well as osteoblast differentiation. A common way to coat surfaces is the adsorption method. An alternative way is the use of a protein immobilization method like p-nitrophenyl chloroformate. In this study, we investigated the effect of a collagen type I coating and p-nitrophenyl chloroformate as a protein immobilization method on osteoblast adhesion, proliferation, and differentiation. Titanium fiber meshes were treated with sodium hydroxide (NaOH), followed by p-nitrophenyl chloroformate, and coated with collagen type I. Osteoblast-like cells were seeded into the meshes and cultured for 24 days. The cell attachment, proliferation, and differentiation were measured by using Live and Dead assay, cell counting, DNA analysis, alkaline phosphatase activity assay, calcium content measurement, Real Time PCR (QPCR), and scanning electron microscopy (SEM). Results demonstrated that initially less cells were attached to the covalently bounded collagen meshes (NPC-Col) compared with titanium as control (Ti) and adsorbed collagen meshes (ABS-Col). Further, a decreased growth curve of cells cultured on the NPC-Col meshes was observed in comparison with Ti and ABS-Col meshes. The calcium measurements and SEM pictures revealed that all three surfaces showed differentiation of osteoblast-like cells after 8-24 days. On the basis of our results, we conclude that initially less cells were attached to the NPC-Col meshes and that they had a decreased proliferation rate. Further, we conclude that an adsorbed collagen type I coating stimulated the osteoblastic differentiation of rat bone marrow cells.

A Versatile Method for Direct and Covalent Immobilization of DNA and Proteins on Biochips

A Versatile Method for Direct and Covalent Immobilization of DNA and Proteins on Biochips

A Versatile Method for Direct and Covalent Immobilization of DNA and Proteins on Biochips

(Figure Presented) Stuck on sensing: A method is presented for the immobilization of aryl diazonium modified biomolecules by direct electro-grafting onto the surface of a conducting material to create effective on-chip sensing layers (see picture). Proof of concept is given for both proteins (antibodies, enzymes) and DNA

General Method for Site-Specific Protein Immobilization by Staudinger Ligation

Protein microarrays are playing an increasingly important role in the discovery and characterization of protein-ligand interactions. The uniform orientation conferred by site-specific immobilization is a demonstrable advantage in using such microarrays. Here, we report on a general strategy for fabricating gold surfaces displaying a protein in a uniform orientation. An azido group was installed at the C-terminus of a model protein, bovine pancreatic ribonuclease, by using the method of expressed protein ligation and a synthetic bifunctional reagent. This azido protein was immobilized by Staudinger ligation to a phosphinothioester-displaying self-assembled monolayer on a gold surface. Immobilization proceeded rapidly and selectively via the azido group. The immobilized enzyme retained its catalytic activity and was able to bind to its natural ligand, the ribonuclease inhibitor protein. This strategy provides a general means to fabricate microarrays displaying proteins in a uniform orientation.

Characterization of a proton pumping transmembrane protein incorporated into a supported three-dimensional matrix of proteoliposomes

Surface analytical tools have gained interest in the bioanalytical field during recent years because they offer the possibility of more detailed investigations of biomolecular interactions. To be able to use such tools, the biomolecules of interest must be immobilized to a surface in a functioning way. For small water-soluble biomolecules, the surface immobilization is quite straightforward, but it has been shown to be difficult for large transmembrane proteins. In those cases, the solid surface often has a negative influence on the function of the transmembrane proteins. In this article, we present a new approach for surface immobilization of transmembrane proteins where the proteins were immobilized on a surface in a proteoliposome multilayer structure. The surface-binding events and the structure of the surface-immobilized proteoliposomes were monitored using both the quartz crystal microbalance with dissipation monitoring (QCM-D) and surface plasmon resonance (SPR) techniques. With this multilayer proteoliposome structure, it was possible to detect trypsin digestion of the transmembrane protein proton translocating nicotinamide nucleotide transhydrogenase in real time using SPR. The results from the combined SPR and QCM-D analysis were confirmed by fluorescence microscopy imaging of the multilayer structure and activity measurements of transhydrogenase. These results showed that the activity of transhydrogenase was significantly decreased in the bottom layer, but in the subsequent proteoliposome layers 90% of the activity was retained compared with bulk measurements. These results emphasize the importance of an immobilization strategy where the transmembrane proteins are lifted off the solid surface at the same time as the amount of protein is increased. We consider this new method for surface immobilization of transmembrane proteins to meet these demands and that the method will improve the possibility to use a variety of surface analytical tools for the analysis of interactions involving transmembrane proteins in the future.

Development of a gold nanoparticles based chemiluminescence imaging assay and its application

In this paper, a novel gold nanoparticles based protein immobilization method was designed. Biocomposites of gold nanoparticles and proteins were successfully coated on poly(methyl methacrylate) (PMMA) plates and polystyrene microtiter plates. The proteins could be immobilized on solid materials with high density and better bioactivity. Based on above design, chemiluminescence (CL) imaging assay for determination of H 2O 2 and recombinant human interleukin-6 (rHu IL-6) was developed. The linear range and the loading capability were greatly improved when compared with imaging assay performed with direct proteins immobilization. Under the selected experimental conditions, a linear relationship was obtained between the CL intensity and the concentration of H 2O 2 in the range of 1.0 × 10 −6 to 1.0 × 10 −4 mol L −1, and rHu IL-6 in the range of 2.0–312.0 pg mL −1. The detection limits were 2 × 10 −7 mol L −1 (3 σ) for H 2O 2 and 0.5 pg mL −1 for rHu IL-6 with relative standard deviation of 3.8% for 3.0 × 10 −5 mol L −1 H 2O 2, and 4.4% for 39.0 pg mL −1 rHu IL-6. This method has been applied to the determination of rHu IL-6 in human serum with satisfactory results.

Double-Hexahistidine Tag with High-Affinity Binding for Protein Immobilization, Purification, and Detection on Ni−Nitrilotriacetic Acid Surfaces

There is a particular need in protein analysis and purification for specific, functional, and generic methods of protein immobilization on solid supports. Here we describe a double-hexahistidine (His6) tag sequence, comprising two hexahistidines separated by an 11-amino acid spacer, which shows at least 1 order of magnitude stronger binding to Ni-NTA-modified surfaces than a conventional single-His6 tag or two single-His6 tags at N- and C-termini. Using, as a model, tagged versions of green fluorescent protein (GFP), stable and tight binding of the double-His6 tag/Ni-NTA interaction was demonstrated by competitive elution from Ni-NTA agarose beads, surface plasmon resonance on a Ni-NTA chip, and ELISA in Ni-NTA microwell plates. Protein purification by Ni-NTA chromatography was improved by a 6-8-fold increase in imidazole concentration required for elution, while the dissociation rate of double-His6 GFP from Ni-NTA chips in SPR (BIAcore) was 10 times slower than for single-His6-tagged proteins. ELISA assays and protein microarrays constructed with double-His6 GFP demonstrated greater detection sensitivity with anti-His antibodies and Ni-NTA conjugates. Moreover, the double-His6 tag could serve simultaneously both for protein immobilization and for detection on surfaces. The double-His6 peptide has the potential to be a universal tag for protein immobilization and detection on arrays and single-step purification of proteins from crude mixtures.

INTEGRATION OF NANOPARTICLES WITH PROTEIN MICROARRAYS

A variety of DNA, protein or cell microarray devices and systems have been developed and commercialized. In addition to the biomolecule related analysis, they are also being used for pharmacogenomic research, infectious and genetic disease and cancer diagnostics, and proteomic and cellular analysis.1 Currently, microarray is fabricated on a planar surface; this limits the amount of biomolecules that can be bounded on the surface. In this work, a planar protein microarray chip with nonplanar spot surface was fabricated to enhance the chip performance. A nonplanar spot surface was created by first coating the silica nanoparticles with albumin and depositing them into the patterned microwells. The curve surfaces of the nanoparticles increase the surface area for immobilization of proteins, which helps to enhance the detection sensitivity of the chip. Using this technique, proteins are immobilized onto the nanoparticles before they are deposited onto the chip, and therefore the method of protein immobilization can be customized at each spot. Furthermore, a nonplanar surface promotes the retention of native protein structure better than planar surface.2 The technique developed can be used to produce different types of microarrays, such as DNA, protein and antibody microarrays.

Selective Immobilization of Proteins onto Solid Supports through Split‐Intein‐Mediated Protein Trans‐Splicing

Protein microarrays have emerged as important tools for screening protein-protein interactions and hold great potential for various applications including proteomics research, drug discovery, and diagnostics. This work describes a novel method for the traceless immobilization of proteins to a solid support through split-intein mediated protein trans-splicing. This method has been successfully used for the immobilization of biologically active proteins from very diluted samples ({approx}1{micro}M) and it does not require the purification of the protein to be attached. This makes possible the direct immobilization of proteins from complex mixtures such as cellular lysates and it can also be easily interfaced with cell-free expression systems for high-throughput production of protein microarrays.

Detection and Quantification of Protein Biomarkers from Fewer than 10 Cells

The use of antibody microarrays continues to grow rapidly due to the recent advances in proteomics and automation and the opportunity this combination creates for high throughput multiplexed analysis of protein biomarkers. However, a primary limitation of this technology is the lack of PCR-like amplification methods for proteins. Therefore, to realize the full potential of array-based protein biomarker screening it is necessary to construct assays that can detect and quantify protein biomarkers with very high sensitivity, in the femtomolar range, and from limited sample quantities. We describe here the construction of ultramicroarrays, combining the advantages of microarraying including multiplexing capabilities, higher throughput, and cost savings with the ability to screen very small sample volumes. Antibody ultramicroarrays for the detection of interleukin-6 and prostate-specific antigen (PSA), a widely used biomarker for prostate cancer screening, were constructed. These ultramicroarrays were found to have a high specificity and sensitivity with detection levels using purified proteins in the attomole range. Using these ultramicroarrays, we were able to detect PSA secreted from 100 LNCaP cells in 3 h and from just four LNCaP cells in 24 h. Cellular PSA could also be detected from the lysate of an average of just six cells. This strategy should enable proteomic analysis of materials that are available in very limited quantities such as those collected by laser capture microdissection, neonatal biopsy microspecimens, and forensic samples.

Photonic activation of disulfide bridges achieves oriented protein immobilization on biosensor surfaces

Photonic induced immobilization is a novel technology that results in spatially oriented and spatially localized covalent coupling of biomolecules onto thiol-reactive surfaces. Immobilization using this technology has been achieved for a wide selection of proteins, such as hydrolytic enzymes (lipases/esterases, lysozyme), proteases (human plasminogen), alkaline phosphatase, immunoglobulins' Fab fragment (e.g., antibody against PSA [prostate specific antigen]), Major Histocompability Complex class I protein, pepsin, and trypsin. The reaction mechanism behind the reported new technology involves "photonic activation of disulfide bridges," i.e., light-induced breakage of disulfide bridges in proteins upon UV illumination of nearby aromatic amino acids, resulting in the formation of free, reactive thiol groups that will form covalent bonds with thiol-reactive surfaces (see Fig. 1). Interestingly, the spatial proximity of aromatic residues and disulfide bridges in proteins has been preserved throughout molecular evolution. The new photonic-induced method for immobilization of proteins preserves the native structural and functional properties of the immobilized protein, avoiding the use of one or more chemical/thermal steps. This technology allows for the creation of spatially oriented as well as spatially defined multiprotein/DNA high-density sensor arrays with spot size of 1 microm or less, and has clear potential for biomedical, bioelectronic, nanotechnology, and therapeutic applications.

A new method for immobilization of lipid-binding proteins to the glass slides

Abstract Previously, we constructed nickel-coated glass slides to immobilize histidine-tagged proteins [Bull. Korean Chem. Soc. 23 (2002) 1724–1728]. Here, we further investigated whether the nickel-coated glass slide could be utilized for assaying molecular interaction between lipid and immobilized protein. Proteins interact with various lipid molecules in biological systems. More importantly, this interaction is responsible for a broad spectrum of physiological functions. Accordingly, to more efficiently analyze various types of interactions and understand the complicated biological systems, it is essential to develop a high-throughput analytical device. For the high-throughput analytical purpose, we immobilized an FITC (fluorescein isothiocyanate)-tagged lipid-binding protein to nickel-coated plates, incubated the plates with various lipid molecules and then measured fluorescence intensities to analyze its binding affinity. Our systems seemed to be reliable for the application to the high-throughput system consisted of lipid-binding proteins.