Protein immobilization methods

We have discovered a novel protein immobilization method, i.e., a Three-Dimensional Nanostructured Protein Hydrogel (3-D NPH), which is composed of protein-reactive polymer hybrid nanoparticles to detect protein-protein interactions. The 3-D NPH can be easily prepared by spotting a protein/reactive polymer mixture on a substrate. The resulting 3-D NPH is characterized by large amounts of immobilized proteins and a novel porous structure. 

The 3-D NPH technology was applied to immobilize streptavidin (SA) onto Au-coated surface for surface plasmon resonance imaging (SPRi). By using 3-D NPH method, it was possible to improve the sensitivity of protein-protein interactions drastically comparing to the conventional protein immobilization method. 

Houston B, Peddie D (1989) A method for detecting proteins immobilized on nitrocellulose membranes by in situ derivatization with fluorescein isothiocyanate. Anal Biochem 177 263-267... 

The adsorption of proteins onto surfaces is the oldest and easiest immobilization method. Adsorbing forces can be of different types Van der Waals interactions, ionic, hydrophobic or hydrogen bonding. The main advantages of this procedure are the simplicity of preparation and the little... 

The physical adsorption of protein onto the surface of an electrode is a simple immobilization method. The adsorption is obtained by volatilizing the buffers containing proteins. The physical adsorption needs no chemical reagent, seldom activation and rinse, so that the bioactivities of the immobilized proteins can be retained well. However, the immobilized proteins are easy to break off from the electrode, which restrict broad applications of this method. Below are some examples of the physical adsorption of proteins immobilized on electrodes. 

Suaud-Chagny and Gonon [3] presented a new procedure for protein immobilization adapted to carbon microelectrode characteristics. The principle of this method of immobilization is based on the association of the protein with an inert porous film immobilized around the active tip of the electrode. For this purpose the carbon was coated with an inert, electrochemically obtained protein sheath (bovine serum albumin, BSA) a few micrometers thick. Then the sheath around the fiber was impregnated with lactate dehydrogenase (LDH), which could be immobilized onto the electrode and resulted in an electrode sensitive to pyruvate. 

Covalent immobilization is performed through a chemical reaction between the protein molecule and the solid support (matrix). While the reactive moieties on the support can be chosen relatively freely, chemical modification of the protein tends to result in a decrease of its biological activity. Therefore, immobilization via reactive residues of the amino acids is preferred. To design suitable immobilization methods some guidelines should be followed. 

Immobilization methods based on eovalent eoupling are preferable, as covalent coupling gives a stable immobilization and the receptor do not dissociate from the surface or exchange with other proteins in the solution. 

Figure 6.27 In-well PISA and His tag-expressed protein immobilization. (From He, M. et al., /. Immunol. Methods, 274, 265-270, 2003. With permission.)...

A third item to consider in using affinity chromatography is the way in which the ligand is attached to the solid support, or the immobilization method. Several techniques are available for this, including both covalent and noncovalent coupling methods [25,36]. For a protein or peptide, this generally... 

The adequate protein/polymer concentrations in feed for 3-D NPH exist for SPRi, because the evanescent electromagnetic field decays exponentially from the metal surface into the interacting medium as described above. The thickness of 3-D NPH-SA prepared over the concentration of 0.25/0.025% is too thick to monitor SPR interactions even at the center of spots. It is thought that different proteins have each optimum concentration for 3-D NPH immobilization method in the case of SPR measurements. 

Lee Y, Lee EK, Cho YW, et al. (2003) Pro-teoChip a highly sensitive protein microarray prepared by a novel method of protein immobilization for application of protein-protein interaction studies. Proteomics 3, 2289-304. 

Affinity chromatography (AC) is a fractionation technique widely used in targeted proteomics or protein-protein interaction approaches. This method utilizes an interaction or affinity of a target protein to a substrate (or another protein) immobilized on a support matrix. Figure 17.4 demonstrates that AC can be incorporated into 3D proteomic approaches (Lee and Lee, 2004). Traditionally, AC has been used to purify, for example, carbohydratebinding proteins from Diplostomum pseudo-spathaceum (Mikes and Man, 2003) and the detoxification superfamily glutathione transferase (GST) from parasitic flatworms (Brophy and Barrett, 1990), specifically shown in... 

The avidin-biotin immobilization method maintains the biological activity of the receptor molecules. Besides the biocompatibility of the procedure, the surface geometry of these films provides high accessibility of the immobilized biomolecules. In addition, the avidin molecules form a passivation layer on the transducer surface that prevents nonspecific adsorption of proteins on the surface. In contrast with conventional grafting or affinity binding, this step-by-step approach can also be applied to the preparation of assemblies containing multilayers of biological molecules [42],... 

One of the most important features in the immunosensor design is the proper choice of the immobilization method for keeping the affinity of the antibodies. As was previously demonstrated for Protein A, when the antibodies are immobilized through their Fc fragment to Protein A (or G), their Fab binding sites are mostly oriented away from the solid phase. As Protein A is able to link the Fc region of different antibodies, there is no need to modify the antibody with biotin. As an antecedent, we have previously demonstrated the utility of Protein A biocomposite (ProtA-GEB) for the universal attachment of antibodies with different specificities [54]. 

Surface chemistry is a key technology for protein microarray development. The supports used for protein immobilization have to fulfil some important requirements they must provide good quality spots, low background, simplicity of manipulation and compatibility with detection systems. An ideal surface or immobilization procedure for all proteins and applications does not exist however current methods are more than adequate for many applications. Basic strategies for protein immobilization consider covalent versus non-covalent and oriented versus random attachment, as well as the nature of the surface itself [106]. It has been demonstrated that the specific orientation of immobilized antibody ( capture agents ) consistently increases the analyte-binding capacity of the surfaces, with up to 10-fold improvement over surfaces with randomly oriented capture agents [107]. 

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