Coupled heat-inactivation

Table 5 L18 Hunter Design Table for the Qualification Study for the Coupled Heat-Inactivation/ Precipitation Steps... 

Add 5 ml Antibiotic-Antimycotic (lOOx, cat. 15240-062) and 50 ml heat-inactivated horse serum (cat. 16050-122) to make complete MEM (used for neuron plating and glial culture). For neuron plating, the complete MEM could be kept for a couple of months at 4°. However, for glial culture, a freshly thawed serum aliquot should be used for each culture. Obvious growth retardation in glial culture is observed when using 1-month-old complete MEM stored at 4°. 

The tissue preparations were diluted (1 12) in heat-inactivated sera (1 hour at 55°C), and incubated in 0.02 M Veronal buffer (pH 9.8) containing 0.018 M disodium phenyl phosphate for 2 hours at 37°C. Phenol was measured via a diazo coupling procedure. Conditions for heat-inactivating the tissue enzymes were 16 minutes at 55°C. By subtracting from the total activity the intestinal component, which is measured by n-phenylalanine sensitivity, one obtains the sum of the activities of liver and bone. The ratio of the two was computed from the heat inactivation minus that attributed to intestine, employing 91.2% heat inactivation to represent 100% bone and 51.4% heat inactivation indicating all liver. In this way one arrives at values for bone, liver, and intestinal alkaline phosphatase. 

In the second example, we demonstrated that a process module might be comprised of one or more unit operations. In this example, the process module investigated comprised of several emit operations—a heat inactivation step, a precipitation step, and a filtration step. Depending on the complexity of the steps, it may be beneficial to separate them initially and evaluate their individual characteristics. However, as the steps may be coupled, as was the case in the example provided, a qualification study should evaluate the process module as a whole. In the example provided, we were able to clearly demonstrate the robustness of the module by comparing the output of the module against a subset of the established finished-product specifications. 

Using a simple affinity system which required coupling of a-L-fucopyranose to macroporous agarose by epichlorohydrin treatment of the agarose in the presence of a-L-fucopyranose under alkaline conditions has been used to resolve a partially purified preparation of monkey brain a-L-fucosidase at pH 5.0 into two major fractions one bound and one retarded.The enzyme that was bound to the affinity column and specifically eluted by 2 mM a-L-fucose at pH 5.0 appeared to be homogeneous by polyacrylamide gel electrophoresis and was constituted mainly by the tetrameric form of the enzyme. The enzyme fraction retarded by the affinity column was found to contain mainly the monomeric form of the enzyme. Additional evidence for the different molecular forms of enzyme in the bound and retarded fractions came from pH-activity profiles and heat inactivation studies. The L-fucose-affinity matrix appeared to bind the tetrameric form of the enzyme specifically and, further, a-L-fucopyranose helped to retain the molecular integrity of the tetrameric enzyme. 

Cellular proteins were transferred to nitrocellulose in a Trans-Blot cell (Biorad). The gel was pre-equilibrated in transfer buffer (0.7% acetic acid) for 30 min. The transfer was then performed with reversed polarity at 30 v overnight. The nitrocellulose was incubated in blocking buffer (5% Carnation non-fat dry milk in PBS) at 37 C for 30 min. The nitrocellulose was then washed in PBS-Tween (0.05% Tween-20 in PBS) three times for 10 min. A 25 mL solution of primary antibody (12.5 pL polyglue antiserum, 250 pL heat-inactivated horse serum, 24.7 mL PBS-Tween) was added for 1 h. After washing in PBS-Tween three times for 5 min, the secondary antibody solution (goat anti-rabbit IgG coupled to horseradish peroxidase, Cappel) was added for 0.5 h. The nitrocellulose was then washed in PBS-Tween three times for 5 min and PBS once to remove residual Tween. The blot was developed using diaminobenzidine as enzyme substrate. 

Fig. 8.2 A self-organising system needs a flow of energy. To continuously overcome the loss of entropy due to irreversible processes, it must be coupled to a flow delivering energy in a low entropy form and dissipate it under the form of heat or inactivated chranical derivatives...

The concept of spatial confinement using picosecond laser-heated nanopartides was studied via protein denaturation and subsequent inactivation. In their initial study, Huttman et al. [88] characterized the extent of the spatial confinement by changing the distance between the target and the particle surface. Initially, 15 nm gold nanospheres were directly coupled with the protein alkaline phosphatase (aP). 

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