Cooling incubation time

There are several recent examples of the switching of nonspecific protein binding on polymer surfaces by application of an external stimulus. Alexander and coworkers demonstrated that protein adhesion can be controlled on PNIPAM surface brushes [14, 181]. For instance, it was reported that the adsorption of FITC-labeled bovine serum albumin (FITC-BSA) on PNIPAM/hexadecanethiol micropatterned surfaces could be tuned by LCST. However, this effect was found to be less pronounced after prolonged incubation times or repeated heating/cooling cycles. The authors suggested that this behavior could be due to unspecific PNIPAM-protein interactions [14],... 

FIG. 24 Moffit-Yang plot of the reduced specific rotation of bovine serum albumin (m ) as a function of the wavelength (A) and at different temperature and incubation times. In this representation, the slope of the curve is proportional to the fraction of helices in the secondary structure. Upper diagram mixture of protein and HMPA (2 1 mass ratio). Lower diagram solution of protein alone in a phosphate buffer pH 7. Protein at 0.3 wt%. Key (0) initial solution at 25°C (o) same solution just heated at 70°C ( ) after a 1 h incubation at 70°C ( ) after its cooling back to 25°C. 

A new technique uses microwaves to speed the diffusion of reagents into tissues. Microwaves can be used for ultra-rapid processing of tissue and speeding incubation times. While the theories are still not well understood, they might go something like this Microwaves cause increased vibration of polarized molecules in the tissue and this speeds the diffusion. The microwave has a cooling bath so that the tissue samples stay at room temperature during the exposure. This method is useful for... 

This experiment was carried out in accord with an established procedure (5). Unbleached softwood kraft pulp (USKP) from Douglas fir was obtained from Pope and Talbot (Halsey, Oregon) and washed with deionized water until the effluent was neutral and colorless. USKP (8.0 g, oven-dried) was autoclaved at 121 °C for 40 min, cooled down at room temperature and then inoculated with 3 mL of P. cinnabarinus spore suspension (ca. 9 x lO spores/mL). The moisture content of the culture was adjusted to 80% and the culture was incubated statically at 30 °C. After a pre-determined incubation time, mycelia were carefully removed fiom pulp. Hie treated pulp was analyzed for kappa number. 

The slides with tissue sections are brought to room temperature before incubation with the radiolabeled ligands in buffer at various temperatures (Table 1). After incubation, the sections are rinsed in buffer, briefly dipped in distilled water to remove salts, and rapidly dried under a stream of cool, dry air to minimize possible diffusion. In biochemical studies, this latter step is unnecessary because the sections are wiped off with a piece of filter paper and counted in a scintillation counter. As discussed above, these preliminary biochemical studies are imperative to assess the receptor and derive proper conditions (incubation times, wash times, and so on) for the autoradiography... 

Using a flamed and cooled loop, transfer a young culture (incubation time of 24 to 48 h) to the medium and incubate at 25°C/77°F for 2 to 3 days prior to examination with a microscope. 

Figure 22 A model for thermosensitive cell attachment, (a) Below the transition temperature the surface resists protein adsorption and cell attachment, (b) Raising the temperature above the transition temperature allows adsorption of serum proteins and cell attachment, (c) During extended cell culture, secretion of ECM proteins along with cell proliferation and communication results in confluent cell sheets, (d) Low seeding density or short cell incubation times results in adherent cells without extensive cell-cell junction formation, (e) and (f) Cooling to below transition temperature allows release of intact cells or cell sheets along with associated ECM proteins. Reprinted from Cole, M. A. Voeicker, N. H. Thissen, H. Griesser, H. J. Biomaterials2009,30, 1827-1850, with permission from Elsevier.

If the incubation temperature is increased, the rate of color formation increases and the total amount of color produced by a given mass of protein increases. After cooling the reaction mixture back to room temperature, the rate of color development slows from its initial rate. As both time and temperature are increased, the total amount of color produced by a given mass of protein approaches a maximum. This is ap-... 

Free-floating sections (40 xm) of paraformaldehyde-fixed tissues are rinsed three times for 5 min each in 0.1 M sodium phosphate buffer (pH 7.4) (Jiao et al., 1999). They are transferred to 10-15 mM sodium citrate buffer (pH 8.5-9.0) preheated in a water bath kept in a conventional oven at 80°C for 30 min. The sections are allowed to remain in this buffer for 30 min to cool to room temperature. Following rinsing three times for 5 min each in the same buffer, the sections are treated by immersion in 0.3-3% nonfat dry milk in 0.1 % sodium azide for 30-60 min. The sections are then incubated in the primary antibody, diluted with a mixture of 0.3% Triton X-100, 0.01% sodium azide, 0.1 M sodium phosphate buffer (pH 7.4) (PBX), and 5% normal horse serum for 72 hr at 4°C under constant agitation. 

The following procedure is more suitable for routine application than other methods as many as 200 specimens can be processed at a time with this procedure. Sections (2 pm thick) of formalin-fixed and paraffin-embedded tissues are mounted on silane-coated slides. They are deparaffmized with xylene and rehydrated in a series of descending concentrations of ethanol. The sections are immersed in 0.01 M sodium citrate buffer (pH 6.0) in plastic Coplin jars and heated in an autoclave at 120°C for 20min. After the sections have cooled down to room temperature for 20 min, they are incubated in the freshly prepared following silver staining solution for 25 min at room temperature. 

Dissipation in stream water with sediment In a concurrent study, a series of 120, 100 g aliquots of coarsely sifted stream sediment were placed in 500 mL Erlenmeyer flasks containing 200 mL of stream water each. One half of the samples, i.e., 60 flasks, were autoclaved as before in an Ameco sterilizer for 1 h. After they were cooled to room temperature, all samples including the 60 non-autoclaved samples were separated into two sets (20 autoclaved + 20 non-autoc laved for each set) and one set was fortified with aminocarb and the other with fenitrothion in acetone to a level of 100 ppb (30 yg/ 300 g) and incubated in an environmental chamber as described above. The remaining 40 flasks served as controls for both experiments. Samples of both the autoclaved and the non-autoclaved water and sediment in open and closed flasks as well as control samples were analyzed for the active ingredients 1.0 h after fortification (zero time) and thereafter at intervals of time up to 75 h.. 

The medium is cooled and inoculated with 20 ml of a suspension of the spores from two Mover s sporulation agar slant cultures of Streptomyces rimosus forma paromomycinus in sterile 0.1% sodium heptadecyl sulfate solution. The inoculated culture mixture is incubated at 26°C for sixty hours during which time the mixture is stirred at 200 rpm and sterile air is passed into the medium through the sparger at the rate of 12 liters per minute. A portion of the resulting incubated culture mixture is employed for inoculation of 16 liters of a nutrient medium having the following composition ... 

---