Methods Cell homogenates

Most of these methods are suitable for analyzing the components present in cell homogenates and other biochemical preparations. The sequential use of several techniques will generally permit purification of most biomolecules. The reader is referred to texts on methods of biochemical research for details. 

With the exception of gel filtration, all modes of biochromatography are, in principle, adaptable to EBA. Separations based on ion exchange [27], Protein A affinity [28] and IMAC [29] have been published. The list of feed includes bacterial fermentation broth [30], cell homogenate [31] and renatured inclusion bodies [32], yeast fermentation broth [29] and cell homogenate [27] as well as mammalian or hybridoma cell culture broth [33]. EBA is also an accepted method in the production of medicinal products. Although, considering the problems in initial processing discussed earlier... 

Most organelle membranes, such as the tonoplast (6) and the Golgi apparatus (7), can be separated by density gradient ultracentrifugation of plant cell homogenates. However, other effective methods for the isolation of the plasma membrane (8,9) have been described. Moreover, another method that uses an aqueous two-phase system for the isolation of ER is also described (10). Those interested in these details for these methods should consult the original articles. 

The preparation of the sample prior to its analysis will depend upon the nature of both the sample and the analytical method chosen and may involve the disruption of cells, homogenization and extraction procedures as well as the removal of protein or other interfering substances. It may be necessary to prevent the decomposition and degradation of the carbohydrate content during such treatments or during storage by the addition of antibacterial agents such as thymol or merthiolate, or substances such as fluoride ions, which will inhibit the enzymic transformation of the carbohydrates. 

The method used to determine DNA concentration will depend on the nature of the sample. In recombinant DNA technology relatively pure samples of DNA or RNA are used and so simple spectrophotometric methods are adequate. However, when there are large amounts of interfering substances present, such as in a tissue or cell homogenate, a chemical method will be necessary. 

After cell disruption, gross fractionation of the properly stabilized, crude cell homogenate may be achieved by physical methods, specifically centrifugation. Figure 7.11, Chapter 7, outlines the stepwise procedure commonly used to separate subcellular organelles such as nuclei, mitochondria, lysosomes, and microsomes. 

For solid materials, it is first necessary to homogenize the sample. This can be accomplished only after the physical structure of the sample is destroyed. This can be as simple as grinding seeds in a mortar and pestle or using a food blender to create a puree from a sports nutritional bar. Additionally, one may wish to sonicate the sample to rupture cells and release their content. A review that also discusses more stringent methods for homogenization (e.g., hammer milling, C02 milling) is presented by Lichon (6). 

Thus, many effective methods for PolyP determination and characterization have now been developed. These allow PolyP assays not only in extracts, but also in cell homogenates or even in living cells. The most appropriate information about the PolyP content, polymerization degree and metabolism may be obtained by combining different methods, including the extraction of PolyPs from cells. 

Activity Determination (Day 6) The cells are harvested for the evaluation of enzyme activities. This can be performed in situ via the incubation of the cells with the enzyme substrates (Table 2). Activity can also be determined from the cell homogenates or microsomal fractions of the cells. The in situ method is the most efficient, and would allow one to use 24-well or 96-well plates. The microsomal method would require the use of larger cell culture plates (e.g. 100-mmdiameterplates). DME substrates used in the hepatocyte enzyme induction assay are shown in Table 4. 

The in situ method for enzyme activity measurement is the most efficient. Enzyme activity can also be measured from cell homogenates or microsomal preparations. Besides activity, one can also measure mRNA and protein levels. 

The most reliable method for assessing thiamin status involves the measurement of red blood cell transketolase. This enzyme is measured with and without the addition of TPP to the enzyme assay mixtures. In dietary thiamin deficiency, synthesis of transketolasc continues, but conversion of the apoet zyme to the holoenzyme in the cell is inhibited, resulting in the accumulation of the enzyme in the apoenzyme form. Addition of TPP to cell homogenates results in the conversion of apoenzyme to holoenzyme. This conversion can easily be detected by enzyme assays. The amount of shmulation of enzyme activity by the added TPP is used to assess thiamin status. A deficiency is indicated by a shmulation of over 20%, The TPP-dependent stimulation, using red blood cells from normal subjects, ranges from 0 to 15%. 

The Lowry method was used to determine the protein concentration in brain and tumour cell homogenates. The method was found to be reproducible for both preparations and for different dilutions of the preparations. The... 

Differential Centrifugation The most common initial step in protein purification is the separation of soluble proteins from Insoluble cellular material by differential centrifugation. A starting mixture, commonly a cell homogenate, is poured into a tube and spun at a rotor speed and for a period of time that forces cell organelles such as nuclei to collect as a pellet at the bottom the soluble proteins remain in the supernatant (Figure 3-3la). The supernatant fraction then is poured off and can be subjected to other purification methods to separate the many different proteins that it contains. 

Removal of cell debris, DNA and proteases, constitute the major problems faced during the large-scale isolation of intracellular proteins. This has emphasized the need for rapid prodedures for protein purification. The fundamental studies on separations in aqueous two-phase systems suggest that it is possible to spontaneously partition the protein of interest into one of the phases by selecting the suitable conditions. The method is very efficient and convenient when set up. However, it may be very laborious to find the proper conditions for a favourable partitioning. Furthermore, such a system may be influenced by variations in parameters such as composition of the cell homogenate. Several examples on large scale isolation of proteins have been reported. Some of these are listed in Table II. 

The systems are economically acceptable when separating proteins out of a cell homogenate. On the other hand, when carrying out bioconversion processes, the phase system has to be continuously reused, hence, there must be a method to remove the products from the phase system. This can be performed in a number of ways like adsorption, membrane filtration, etc. 

Biochemical methods can be tuned to achieve the highest selectivity for the manipulation of cells from complex cell suspensions. Cell patterning (see Cell Patterning on Chip ) is now a well-established method and homogeneous as well as heterogeneous patterns, with various numbers of cells and, in various configurations, have been described [2]. Selective... 

---