Reagents incubation

As phenolics are well-known antioxidant agents, the Folin-Ciocalteau total phenolics assay was performed to assess phenolic content in ginseng roots. This assay was performed as described by Singleton et al. [20] with modifications [21]. Briefly, extract dissolved in 70% methanol was combined with Folin-Ciocalteau reagent, incubated for 5 min, and then 7.5% NaHCOs was added. Samples were transferred into microplate wells in triplicate, incubated in darkness for 2 h, and then absorbance read at 725 nm. Samples were blanked against a treatment with only solvent. A standard curve of quercetin was produced and data were expressed as quercetin equivalents. 

Secondary or link antibody and /or tertiary reagents too concentrated. Repeat staining. Determine correct concentration for each reagent. Incubation temperature and incubation time will affect results. To determine optimal incubation protocol, vary both the time and temperature for each reagent in the IHC staining protocol. 11-13... 

After mixing the reagents, incubation was carried out at +4° C for 3 days. The bound fractions were then sedimented by centrifugation for 30 min at 3000 rpm (about 2200 g) at +4 °C. The supernatant phases were eliminated by inverting the tubes which were then allowed to stand on cotton-wool for 30 min. 

Having established that our assay protocol permitted measurement of the specific binding of pH]LPA to GPR23, we examined next the ability of unlabeled LPA to displace [3H]LPA from the receptor. Different concentrations of unlabeled LPA (1 pM to 3 pM) were added to each well, followed by 20 pL of premixed beads and membrane. After incubation at room temperature for 30 min, 10 pL of [3H]LPA (final concentration 20 nM) were added and all reagents incubated at room temperature for 2 hr in the dark prior to reading. We found that unlabeled LPA displaced [3H]LPA in a concentration-dependent manner with a calculated inhibitory constant (K ) of 14 nM (Figure 4.2D). 

The TRF assay design can be reduced to basically a 6 step protocol using a coated microtitration plate and a separation step i.e. add reagents, incubate, wash X 2, measure. With the advent of simultaneous 96-well microtitration plate washers it only takes a few seconds to wash an entire microtitration plate two or three times. Thus use of homogeneous or non-separation assays do not necessarily improve assay times. It does however allow the measurement of reactions where components have weak binding affinity where washing would be detrimental. 

A type of automatic analysis in which samples are processed in separate reaction tubes. A typical discrete analysis system consists of a series of reaction tubes into which the sample is dispensed together with reagent. Incubation or addition of further reagents can follow and then the optical density can be measured by direct reading or by drawing the contents of the... 

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