Incubation pulses

Poly(methyl methacrylate) (PMMA) exposed to radiation at 248-nm, 13-ns pulses (FWHM) and a fluence of 0.4 or 0.8 mJ/cm2 is an example of a system that exhibits the requirement of incubation pulses prior to the onset of ablation.28 Sufficient photon-material interaction is present for the ablation of good absorbing polymers thus, incubation is typically not observed and is not pertinent for the remainder of this discussion. 

At the end of the incubation pulse, spin the tube to pellet the immunocomplex. 

Figure 5. Selected HPLC elution profile of products obtained after incubation of 0.25% polygalacturonate with PGII, upper trace, and PGII H223A, lower trace, respectively, demonstrating the effect of the mutation on catalysis. G1 to G3 indicate the peaks of the corresponding oligogalacturonates. IS indicates the internal standard, glucuronate. The vertical axis shows the pulsed amperometric detector response while the horizontal axis shows the retention time.

HPAEC analyses were carried out to determine the oligomeric products released from various pectic substrates after depolymerization by the PL isoenzymes. Action pattern analyses for the concerted action of PL isoenzymes utilized 68% esterified pectin as substrate. One-ml reaction mixtures in a buffer system as detailed in section 2.2. comprising 0.5% (w/v) substrate and 5 U of enzyme were incubated for 30 s to 18 h, and then thermoinactivated. Samples of 750 pi were applied to a Carbopac PA-1 (Dionex) column before the carbohydrates were eluted over a period of 70 min using a gradient of 0.2 M KOH, 0.05 M K-acetate to 0.2 M KOH, 0.7 M K-acetate. Detection employed a Pulsed Electrochemical Detector (PED, Dionex) in the integrated amperometry mode according to the manufacturer s recommendations. 

Fig. 5.5. Percentage of GnRH self-priming in hemipituitaries from ovariectomized rats treated for 3 d with oil, B, or TX and incubated for 3 h with medium alone, E2, or TX. GnRH self-priming was calculated as the peak response to the second GnRH pulse x 100/peak response to the first GnRH pulse. A value of 100% or less indicates absence of GnRH selfpriming. P < 0.05 versus oil (modified from Sanchez-Criado et al. J Endocrinol 186 43-49, 2005)...

The biosynthesis of myeloperoxidase has been characterised by pulse-chase experiments. This is achieved by incubating cells with a radioactive... 

Figure 7 Immunopotentiating reconstituted influenza virosomes (IRIV) mediated adjuvance in cytotoxic T-cell induction requires CD4+ T cells. CD8+ and CD14+ cells were cultured in the presence of autologous intact or irradiated CD4+ cells. These cultures were stimulated with influenza matrix (IM)58 66 (1 Pg/mL) alone (A) or supplemented with IRIV (1 50) (B). After seven days of incubation both cocultures were restimulated with irradiated IMss-ee pulsed CD14+ cells and cultured for six further days in the presence of interleukin-2 [see Materials and Methods ]. Six days after restimulation, cultures were stained with HLA-A0201 /IM58-66 PE-specilic tetramers and anti-CD8 fluorescein isothiocyanate monoclonal antibodies. Source. From Ref 6.

N]-, [ C]-, pHjleucine or p Sjmethionine in the case of proteins) for various periods, after which the cells are lysed and the protein of interest is purified (often by immunoprecipitation with specific antibodies). The time course of isotope incorporation gives information about the rate (slope of curve) and extent (amplitude of curve) of the proteins synthesis. To measure degradation, cells are first pulse-labeled (i.e., exposed to radiolabeled precursor for a fixed period, after which sufficient nonla-beled precursor is added to reduce the radiospecific activity of the precursor). Then, the cells are further incubated, and the radiospecific activity of a particular protein of interest is determined (again usually after immunoprecipitation or some other means for achieving its isolation from other cellular proteins). The key point is that the chase allows one to stop radiolabel uptake almost instantaneously, thereby permitting the kinetic... 

Alice et al studied the turnover kinetics of Listeria OTonocytogenex-secreted p60 protein (a murein hydrolase) by host cell cytosolic proteasomes. J774 cells, seeded in flasks and incubated overnight in culture medium, were infected with log-phase cultures of E. monocytogenes for 30 min, washed, and incubated in culture medium for 3 h, with gentamicin (50 tg/ml) added after the first 30 min to inhibit extracellular bacterial growth. Cells then were washed and placed in methionine-free medium with spectinomycin, gentamicin, the eukaryotic protein synthesis inhibitors [cycloheximide (50 tg/mL) and anisomycin (30 tg/ml),] and 25 dVI calpain inhibitor I. After 30 min, [ S]methionine was added, and the cells were pulse-labeled for periods of 20 to 60 min. Cells... 

Three microliters of the donor DNA solution and 1 pL of the trap vector solution are blended with a 20 pL of competent JC8679 cells for electroporation see Note 10), and the mixture is transferred into an electroporation cuvette with a 0.1 cm electrode gap. After an electric pulse of 1.67 kV is applied, 1 mL of SOC medium is added to the suspension, and incubated at 37°C for 3 h with vigorous shaking. This process is applied to each gene one by one. 

BrdU/DNA flow cytometry offers flexibility and diversity in the study of cell kinetics from cells in culture to human tumors in vivo. The essence of the procedure is to pulse label with BrdU by a short-term incubation in vitro or by a single injection in vivo samples are then taken at time intervals thereafter and stained after fixation in ethanol. The cells are then stained with a monoclonal antibody against BrdU that can be either directly conjugated to a fluoro-chrome (usually fluorescein isothiocyanate [FITC]) or, alternatively, bound to a second antibody conjugated with FITC. The cells are then counterstained with propidium iodide (PI) to measure the DNA content and analyzed on the flow cytometer. The results are displayed as linear-red fluorescence on the x-axis vs linear or log-green fluorescence on they-axis. 

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