Proteins Treated Surfaces

The ProteinChip System from Ciphergen Biosystems uses patented SELDI (Surface-Enhanced Laser Desorption/Ionization) ProteinChip technology to rapidly perform the separation, detection, and analysis of proteins at the femtomole level directly from biological samples. ProteinChip Systems use ProteinChip Arrays which contain chemically (cationic, anionic, hydrophobic, hydrophilic, etc.) or biochemically (antibody, receptor, DNA, etc.) treated surfaces for specific interaction with proteins of interest. Selected washes create on-chip, high-resolution protein maps. This protein mass profile, or reten-tate map of the proteins bound to each of the ProteinChip Array surfaces, is quantitatively detected in minutes by the ProteinChip Reader. 

In a more modified approach, differential display proteomics can also be done with no separation of proteins. This is called the protein chip approach. In this method, a variety of bait proteins such as antibodies, peptides, or protein fragments may be immobilized in an array format on specially treated surfaces. The surface is then probed with the samples of interest. Proteins that bind to the relevant target can then be analyzed by direct MALDI readout of the bound material (Nelson, 1997 Davies et ah, 1999). Lor example, well-characterized antibodies can be used as bait. Protein samples from two different cell states are then labeled by different fluorophores, mixed together, and used as probe. In such a case, the fluorescent color acts as an indicator for any change in the abundance of the protein that remains bound to the chip (Lueking et ah, 1999). A number of technical problems would still need to be overcome before applying this technique for large-scale analysis of proteins. 

The most useful information on the interactions of proteins with surfaces will come from studies analogous to those of protein chromatography, where well-characterized and understood proteins are studied with well-characterized surfaces of known functional group type and density. The information obtained is then analyz-able in such a way as to deduce interaction site densities and interaction energies. Only with such data in hand will we be able to begin to quantitatively treat and understand protein adsorption. 

When a polymer is treated with enzymes for surface modification, some of the undesired protein tends to adsorb on the polymer surface, which subsequently creates problems in the surface analysis and causes a slow down in the rate of catalysis. Adsorbed proteins can be removed from the surfaces by washing with large volumes of 1.5% Na2C03 and water (Eischer-Colbrie et al., 2006) as part of a preparation for surface analysis. Protein-resistant molecules such as polyethylene glycol can be used to prevent the nonspecific protein adsorption. Surfaces can be precoated with an inert protein such as bovine serum albumin (Salisbury et al., 2002) for increasing the rate of catalysis. 

The titer reduction and adsorption capacities of the T2 phage and HSV-1 are compared in Table 3. For similar initial titers (10 PFU/ml), the survivor titer of HSV-1 was at least 2 orders of magnitude lower than that of T2. For similar equilibrium titer remaining in the solution (10 PFU/ml), the adsorption capacity (PFU/ml) of HSV-1 was 2 orders of magnitude higher than that of T2. Evidently, HSV-1 is much more susceptible to the surface-bonded QAC than T2. Since HSV-1 is an enveloped virus, the lipid bilayer surroimding the capsid binds strongly to the QAC-treated surface due to additional hydrophobic interaction. It should be noted that the adsorption experiments of T2 were carried out in buffer solutions without proteins, while those of HSV-1 were in buffered 1 vol% FBS solution. 

Lyophilization of the treated surfaces and of removed particles is achieved by the addition of various polymers, such as, e.g., carboxymethylcellulose, which is introduced into synthetic detergent formulations in the amount of a few percent. Detergents also often contain enzymes that are capable of cleaving proteins present in soiled areas. It became possible for one to use enzymes only after the methods of their encapsulation were developed encapsulation of enzymes prevented their degradation by other components present in synthetic detergents. 

It must be stressed that the above findings concerning relative sizes of filled and empty droplets may not be true for other systems. When the protein is surface-active, which is the case for the most frequently employed enzyme, lipase, the model cannot readily be used, because the protein does not reside in the droplet core. Furthermore, the model treats all ions as point charges hence, specific ion effects cannot be accounted for. 

The advantage of the latter reactions is that they are generally applicable to a variety of proteins, synthetic polymers and plasma-treated surfaces [82]. The overall surface density of the peptides can also be eontrolled easily via these chemical modification strategies, which can have a large impaet on eell proliferation and differentiation, particularly when coupled with manipulation of the chemical composition of the matrix material [85]. 

In this regard, plasma treatment is an effective and economical tool in the field of surface modification, which may be used quickly, easily and it does not require relatively expensive devices for its operation. The primary effect of plasma treatment is to convey reactivity to the treated surfaces via electrons, ions and UV-radiation confining the treatment to the top layer without affecting bulk properties. For these reason, plasma surface modification has been done on different materials, such as polymers, carbon fibres, ceramics, and proteins [7-9]. 

The construction of chemically modified solid supports is of particular inportance in cell-based microarrays. For cell immobilisation on substrates the most commonly used method is the binding to poly-L-lysine-coated slides. Binding of cells results from ionic interactions between the cell membrane and the positively charged poly-L-lysine surface. This method is most effective for adherent cell types. To promote cell adhesion, surfaces coated with extracellular matrix (ECM) proteins such as collagen or fibronectin have also been employed. In some cases, polymer-treated surfaces are used in conjunction with the above mentioned compounds to reduce non-specific adhesion. 

Widmer and Spencer [9] treated UHMWPE with oxygen plasma. The treated surface was hydrophilic and had a faster and modified protein adsorption. A denser boundary layer of human serum albumin protein was found on the... 

High concentrations of protein in the medium can inhibit cells from adhering tightly to polylysine-coated surfaces. If this is a problem, allow cells to adhere to the treated surface in serum-free medium for 1-2 hours, and then replace medium with the normal growth medium. 

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