Immobilized proteins

A. Walhagen and F.-E. Edholm, Coupled-column cliromatography of immobilized protein phases for direct separation and determination of dmg enantiomers in plasma , 7. Chromatogr. 473 371-379 (1989). 

Affinity microparticles (AMPs) were obtained by cross-linking the S-layer lattice on S-layer-carrying cell wall fragments with glutaraldehyde, reducing Schiff bases with sodium borohydride, and immobilizing protein A as an IgG-specific ligand [92]. Thus, AMPs rep-... 

Another approach has been to immobilize proteins within arrays of microfabricated polyacrylamide gel pads (Arenkov et al., 2000). Nanoliters of protein solutions are transferred to 100 x 100 x 20-pM gel pads and assayed with antibodies that are labeled with a fluorescent tag. Antigen imbedded in the gel pads can be detected with high sensitivity and specificity (Arenkov et al., 2000). Furthermore, enzymes such as alkaline phosphatase can be immobilized in the gel pads and enzymatic activity is readily detected upon the addition of an indicator substrate. The main advantage of the use of the threedimensional gel pad for fixation of proteins is the large capacity for immobilized molecules. In addition, the pads in the array are separated from one another by a hydrophobic surface. Thus, each pad behaves as a small test tube for assay of protein-protein interactions and enzymatic reactions (Arenkov et al., 2000). The disadvantage of the method is the need to microfabricate the array of gel pads in that microfabrication is... 

The experiments described above indicate that technology is available to couple SPR with mass spectrometry. These methods should be useful for protein-protein interaction mapping. For example, immobilized proteins can be used as hooks for fishing binding partners from complex protein mixtures under native conditions. The coupling of techniques can lead not only to the rapid identification of interacting proteins but will also provide information on the kinetic parameters of the interaction. This approach should serve as an excellent complement to the use of in vivo techniques such as the yeast two-hybrid system. 

Transition-metal phosphorus trichalcogenides such as MnPS3 are able to intercalate amino acids and peptides by ion exchange. In this way, increases in the basal spacing of 0.7 and 3-4 nm are observed for the intercalation of poly-L-lysine and lysozyme, respectively [224]. Interestingly, the enzymatic activity of the immobilized protein has been detected, suggesting that the enzyme is protected against denaturation. 

Weetall, H.H. (1993) Preparation of immobilized proteins covalently coupled through silane coupling agents to inorganic supports. Applied Biochemistry and Biotechnology, 41, 157-188. 

Calcined [MgAl] LDH was also used to adsorb penicillin G acylase [121]. The calcined LDH phases have porous structures, large specific surface areas and abundant basic sites to bind the enzymes. The effect of varying the composition of the LDH precursor and calcination temperature on the activity of the immobilized enzyme has been reported. In this case, the percentage of immobilized proteins increases up to 88 %. 

Vijayendran R.A., Leckband D.E., A quantitative assessment of heterogeneity for surfcae-immobilized proteins, Anal Chem. 2001 73 471-480. 

Apply the fragmented IgG solution to an immobilized protein A column containing 2ml of gel (Thermo Fisher) that was previously equilibrated with lOmM Tris-HCl, pH 8.0. 

Apply the supernatant liquid to an immobilized protein A column (2ml gel Thermo Fisher) which was previously equilibrated by washing with 20ml of lOmM Tris-HCl buffer, pH 8.0. 

Elute Fc and undigested IgG bound to the immobilized protein A column with 0.1M glycine-HCl buffer, pH 2.8. 

No matter what kind of biochip is chosen for use, the first and sometimes the most important step to construct a biochip is to effectively immobilize proteins or biomolecules on a solid surface. The ideal properties of protein biochip surface are ... 

Orientation of immobilized protein with binding site towards the solution. 

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. 

Protein is immobilized by combining with the surface of the electrode through a covalent bond, which is called covalent bonding of protein. The process requires low temperature (0°C), low ion intensity, and physiological pH conditions. Although covalent bonding onto the surface of an electrode is more difficult than adsorption, it can provide a more stable immobilized protein. 

So far, the prevalent method to immobilize protein is sol-gel/polymer embedment of protein. It usually embeds and immobilizes protein in a three-dimensional netlike structure of macromolecule polymer. This technology has some characteristics such as mild conditions, multifarious sol-gels, and controllable membrane aperture and figure. It can also retain the bioactivity of protein well when applied to a high concentration of protein. However, the aggregation process is difficult to control. 

Clays are usually cation-exchangeable aluminosilicates, and exfoliated clay particles have a platelet shape with nanoscopic size. Cast protein-clay films on electrodes have been used to immobilize proteins. The Clay/Mb electrode has good electrocatalytic properties for the reduction of oxygen and hydrogen peroxide [236] and the biosensors can also be made based on these properties. 

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