Amount of Proteins

Several methods have been discussed that are used to identify proteins and determine their primary, secondary, and 3D structure to understand their roles in proteomics. However, in proteomics, it is required to know the amount of different proteins in addition to knowing their nature and structure. There are many ways to determine the amount of proteins in a sample. Most of these methods include colorimetric methods, such as the Lowry or Bradford s test and the spectrophotometric method the latter method determines the protein content based on the amount of UV absorption at 280 nm of light. These methods determine the total amount of proteins in a sample. These methods do not provide the relative amounts of different proteins present in a sample. These methods, therefore, are of no use in the high-throughput analysis involved in proteomics. In proteomics, it is important to know the relative abundance of different proteins, because the proteome varies with the state of the cell and in response to changes in the environment. Protein contents also vary from one cell type to another in 

The amount of proteins present in a large number band separated on 2D gel can be measured, and their relative abundance can be established by a variety of methods. First, the different samples of proteins are separated on 2D gel, and then the intensity of protein bands is measured by the intensity of dyes used to visualize the bands. The intensity of protein bands can be measured by a densitometric scan. Staining with silver stain is sensitive. Staining with certain fluorescent dye is equally useful. Alternatively, proteins in two cell types are labeled by growing cells in the presence of radioactive amino acids, such as methionine containing S35 sulfur. The protein samples obtained from the two cell types are then separated by 2D gel. The protein bands are visualized as spots on an X-ray film placed on the gel. The intensity of each spot on the film is determined by densitometry. 

In this method, protein samples from two different cell types are obtained, and one sample is stained with Cy3 flour (giving red color), whereas the protein in the other sample is stained with Cy5 flour (giving green color). The two samples are mixed and then run on a 2D gel together. After separation, protein bands are visualized. A particular band containing an equal amount of proteins with Cy3 and Cy5 flours is visualized as a yellow spot. However, if in a band there is a several-fold difference of a protein, then these proteins appear as a red or green spot. Those with excess of Cy3 flour 

Protein microarray has been useful in examining the relative abundance of a large number of proteins from two different cell types or from a cell type grown under two different condition. The ability to visualize these differences in a large number of proteins from two cell types at the same time makes this method ideally suitable for proteomic analyses. 

Microarray consists of a microscope glass slide exposed to a protein sample on which many antibodies are individually placed at fixed locations. This slide is then exposed to a protein solution. The spots showing protein-protein interactions are detected and used to identify a large number of proteins and their amount on a single slide. Comparisons of the intensity of spots on different slides exposed to protein solutions from different cell types or from a cell type grown under different conditions reveal the relative 

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Several of the products discussed herein are under intense development. One product, based on recombinant hemoglobin, is in early human trials as of this writing. Other hemoglobin-based solutions are also under review at the EDA. Replacement of red blood cells using massive amounts of protein, free in solution, is an unprecedented therapeutic adventure. 

While the above discussion centered on the rate of disruption, the objective is usually to attain at least 90 percent release of the valuable protein from the cells. Cell disruption with protein solubilization is considered to be first order in amount of protein remaining [Currie et al., Biotechnol. Bioeng., 14, 725 (1972)] ... 

Because the separation is nearly independent of the total amount of protein loaded onto the column, no limitation with respect to the protein concentration exists for concentrations at least up to 60 mg. The high mechanical stability enables the injection of even highly viscous samples with high concentrations of protein. 

Protein is an important component of most foods. Nearly everything we eat contains at least a small amount of protein. Lean meats and vegetables such as peas and beans are particularly rich in protein. In our digestive system, proteins are broken down into small molecules called a-amino acids. These molecules can then be reassembled in cells to form other proteins required by the body. 

Highly active catalysts have been produced by adsorption of lipases onto macroporous acrylate beads, polypropylene particles and phenol-formaldehyde weak anion exchange resins. Protein is bound, presumably essentially as a monolayer, within the pores of the particles. The large surface area of the particles (10m2 g 1) means that substantial amounts of protein can be adsorbed, and the pores are of sufficient size to allow easy access of reactants to this adsorbed protein. 

The degree of saturation of carboxylic CP with protein (Y) is determined by the ratio of the amount of protein bonded under these conditions (at a predetermined concentration in solution) to the maximum amount Y = m/M. In this case, Hill s equation becomes... 

This precipitation process can be carried out rather cleverly on the surface of a reverse phase. If the protein solution is brought into contact with a reversed phase, and the protein has dispersive groups that allow dispersive interactions with the bonded phase, a layer of protein will be adsorbed onto the surface. This is similar to the adsorption of a long chain alcohol on the surface of a reverse phase according to the Langmuir Adsorption Isotherm which has been discussed in an earlier chapter. Now the surface will be covered by a relatively small amount of protein. If, however, the salt concentration is now increased, then the protein already on the surface acts as deposition or seeding sites for the rest of the protein. Removal of the reverse phase will separate the protein from the bulk matrix and the original protein can be recovered from the reverse phase by a separate procedure. 

The rapidly increasing number of novel metagenome-derived genes also led to the identiflcation of major bottlenecks. These are mainly linked to the eflhciency of heterologous gene expression and the amount of protein produced, in particular. 

Comparison of the model-derived DIFF with the experimentally evaluated DIFF show that protein routing can be described in terms of the amount of protein synthesized from catabolic activity that is, in general it is around 20% of the mean carbon flux of the diet. A simple explanation why dp > dw is also proposed. 

Until recently, the possibility that H,K-ATPase consists not only of a catalytic a subunit but also of other subunits was not examined. This was mainly due to the fact that SDS-PAGE of purified gastric H,K-ATPase preparations principally gave one protein band with an apparent molecular mass of about 100 kDa, which was reported to comprise 75% or more of the total amount of protein [6,66,67]. This mass is lower than the mass deduced from its cloned cDNA [40], but may be due to the higher electrophoretic mobility of membrane-bound proteins, as consequence of having relatively high contents of hydrophobic amino acid residues [68]. 

The reactions described so far do not require the involvement of the apo-B protein, neither would they necessarily result in a significant amount of protein modification. However, the peroxyl radical can attack the fatty acid to which it is attached to cause scission of the chain with the concomitant formation of aldehydes such as malondialdehyde and 4-hydroxynonenal (Esterbauer et al., 1991). Indeed, complex mixtures of aldehydes have been detected during the oxidation of LDL and it is clear that they are capable of reacting with lysine residues on the surface of the apo-B molecule to convert the molecule to a ligand for the scavenger receptor (Haberland etal., 1984 Steinbrecher et al., 1989). In addition, the lipid-derived radical may react directly with the protein to cause fragmentation and modification of amino acids. 

We studied the surface pressure area isotherms of PS II core complex at different concentrations of NaCl in the subphase (Fig. 2). Addition of NaCl solution greatly enhanced the stability of monolayer of PS II core complex particles at the air-water interface. The n-A curves at subphases of 100 mM and 200 mM NaCl clearly demonstrated that PS II core complexes can be compressed to a relatively high surface pressure (40mN/m), before the monolayer collapses under our experimental conditions. Moreover, the average particle size calculated from tt-A curves using the total amount of protein complex is about 320 nm. This observation agrees well with the particle size directly observed using atomic force microscopy [8], and indicates that nearly all the protein complexes stay at the water surface and form a well-structured monolayer. 

Deformylation of nascent polypeptides has been shown to be a function essential for growth in E. coli, Staphylococcus aureus and Streptococcus pneumoniae [15-18]. Moreover, antibacterial mode of action studies, using S. pneumoniae or S. aureus strains in which the expression of PDF is controlled by regulatable promoters, have shown that the antibacterial activity of PDF inhibitors is due to their inhibition of the PDF enzyme, as the susceptibility of the strains to these compounds is dependent on the amount of protein present in the cell [19-21]. These results further validate PDF as a target for novel antibiotics. 

As a complement to any (and perhaps all) of the above methods, calorimetry can be utilized in developing an understanding of the overall energetic behavior of the binding event [20]. The overall thermodynamics of any molecular interaction is the sum of both the enthalpic and entropic energy components of the species involved [21]. While these measurements have historically been somewhat limited due to a requirement for a significant amount of protein, new techniques have alleviated the situation substantially [22]. 

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