Overnight incubation

Although several types of fluorescent beads were proposed as a microscopic fluorescence standard 30 years ago,2 beads have not been used as a proteinembedding matrix for routine IHC on FFPE tissue. We recently tested primary coated beads ( Dynabeads, Dynal, New York) that are coated with a goat anti-mouse antibody on the surface of the beads. In the first experiment, a monoclonal antibody to cytokeratin 7 (DAKO, 50pL/34.5pg) was bound to the beads by incubating with the beads (150 pL at a concentration of 109 beads/1 pL) at 4°C in a cold room with an automatic shaker for overnight. Incubation was followed by three phosphate-buffered saline (PBS) washes,... 

The imidazole carbamate group is more stable to hydrolysis in aqueous buffer than the NHS-carbonate group, which is similar in reactivity to an NHS ester. However, this means that the imidazole carbamate also is slower to react and couple with amines. NHS-carbonate reactions usually go to completion within 1-2 hours at room temperature, whereas imidazole carbamates typically require higher pH conditions and overnight incubations to get maximal yield of ligand coupling. 

Fernandez-Patron, C., Castellanos-Serra, L., and Rodriguez, P., BioTechniques, 12 564 (1992). j SYPRO is a trademark of Molecular Probes, Inc., Eugene, OR. k SYPRO Ruby IEF stain requires an overnight incubation. 

Over the years, in addition to developments with ELISA reagents such as labels, there have been improvements in automation. This has enabled ELISA to be utilized as a high-throughput tool. Typically, ELISAs can be performed in several hours to days. The most common practice is to precoat the microtiter plate for an overnight incubation period, with the remainder of the steps performed the following day. While ELISAs are fast when compared to other assays such as bioassays, which can take days to weeks, they might be considered slow when compared to methods like HPLC, in which the time from sample injection to chromatogram is a matter of minutes. 

Figure 1. Inhibition of geranylgeranoic acid-induced apoptotic cell death by a-tocophrol. Increasing concentrations (10 100 pM) of a-tocopherol were added to HuH-7 cell cultures with (closed circle) or without (open circle) 10 pM geranylgeranoic acid. After overnight incubation, the number of the viable cells was counted using a Trypan blue dye exclusion method. Means S.E. (n=3) are shown.

Downstream extraction. The culmre broth was diluted with ethyl acetate and the aqueous phase separated using a separation funnel. The organic layer was collected and dried over anhydrous sodium sulfate. Removal of the solvent by rotary evaporator gave (5)-7-methyl-2-oxepanone as a light yellow oil (6.5 g, 38 % yield). Chiral-phase GC showed 99 % ee and >97 % purity. EI-MS and NMR confirmed the product. Note the unconverted (R)-2-methyl cyclohexanone evaporated completely under the aeration conditions used during the overnight incubation. 

For ligand-gated ion channel receptor expression studies, stage V and VI oocytes are preferred (see earlier). Under a dissecting microscope, oocytes of this stage that are in good condition are sorted from the rest and then incubated at 16-18°C in MBM overnight. Incubation in hypertonic phosphate... 

Incubate the cells at 37°C in the CO2 incubator and take absorbance measurements at 465 nm after the coloured product starts developing. For HeLa, A549, A2058, MCF-7 and Caco2 cells, 2 or 3 h of incubation is sufficient. For Jurkat cells, 18 h (overnight) incubation is required before absorbance measurements should be made. 

The longer the PI is allowed to react with the DNA, the better the staining. Overnight incubation at 4°C is best. 

BA standards methyl esters are prepared by adding 100 pi of 3% anhydrous methanolic hydrochloric acid, to 5-20 pg of each BA and storing them at room temperature for 2 h, followed by solvent evaporation at 55°C under nitrogen. The -butyl esters are prepared by addition of 100 pi of -butanol to 5-20 pg of each BA, followed by addition of 20 pi of a 40% solution of hydrogen chloride in dioxane. The reaction mixtures are heated at 60°C for 4 h, followed by overnight incubation at room temperature, and then evaporated at 60°C under nitrogen. Sil-Prep [hexamethyldisilazane-trimethylchlorosilane-pyridine (3 1 9)] is used for the preparation of TMS ether derivatives of BA esters. The esterified BAs (5-10 pg) are reacted... 

Positively selected cells attached to the beads after incubation can be collected by overnight incubation at 37°C and then application of a magnet to remove the dissociated beads. 

Overnight incubation at 4°C causes beads to dissociate from positively selected cells. However, up to 50% of the selected cells can be lost when magnetic separation is performed, resulting in low yields of bead-free cells. Furthermore, cell viability can be reduced following close contact with the beads. In the author s laboratory, it has been found that positively selected T-cells often do not survive... 

Coat MicroTest III flexible assay plates with protein antigen (100 pL/well) Overnight incubation at room temperature with 10-100 pg/mL of antigen in either PBS, pH 7.2, or 50 mM sodium hydrogen carbonate, pH 9.6, is generally satisfactory. 

Calibration prior to HPLC requires 1 or 2 days to perform all necessary steps (i.e., prepare stock solutions, filter, measure absorbance, dilute, and HPLC injection of the working solutions). Once the stock solution and working solutions have been prepared, a calibration check can be performed on a UV spectrophotometer within 1 hr. The sample clean-up procedure for HPLC analysis may require afew hours. Enzymatic hydrolysis requires overnight incubation. Each HPLC analysis requires 50 min. When an autosampler is available,... 

A third method relies on the precipitation of proanthocyanidins with formaldehyde. First, the total phenolic content is measured using the Folin-Ciocalteu reagent as described before. A 0.5 mole equivalent of phloroglucinol (1.3) is added for every gallic acid equivalent in the extract. To 2 mL of this plant extract and phloroglucinol is added 1 mL of a 2 5 HC1 /H20 solution and 1 mL of an aqueous solution of formaldehyde (13 mL of 37% formaldehyde diluted to 100 mL in water). After an overnight incubation at room temperature, the unprecipitated phenols are estimated in the supematent by the Folin-Ciocalteu method. The precipitate contains the proanthocyanidins and the known amount of phloroglucinol, which is always quantitatively precipitated. 

The sections are treated for 30 min with 10% nonimmune goat serum and then rinsed in PBS. This is followed by overnight incubation at 4°C in monoclonal antibody against... 

Fluid from solid-phase extraction of wool was placed on simple screen-printed electrodes (an outer membrane was applied with an airbrush) [45]. The solvent was allowed to evaporate and, after an overnight incubation, the activity of the electrodes was measured quickly with reference to that of unexposed electrodes. It was possible to detect the presence of organo-phosphates which had been used to contaminate samples of untreated sheep wool (Fig. 28.3) (see Procedure 41 in CD accompanying this book). 

The methods outlined below consist of 1) tube coating with the capture antibody, 2) sample extraction from potato, 3) virus capture in the coated tubes, 4) cDNA synthesis from the captured viruses and 5) PCR amplification of the cDNA. Tube coating requires an overnight incubation, so should be set up on the day prior to extractions. 

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