Big Chemical Encyclopedia

Chemical substances, components, reactions, process design ...

Articles Figures Tables About

Nonspecific duplices

We have found that many compounds identified in our screen are nonspecific inhibitors of luciferase enzyme activity. To eliminate these, we test the hits in a luciferase enzyme-based counterscreen. Firefly and renilla luciferase are produced in vitro by programming Krebs-2 extracts with FF/HCV/Ren mRNA and allowing the translations to proceed at 30° for 1 h. Ten microliters are then pipetted into a 96-well plate and compound is added to a final concentration of 20 /iM (1% DMSO). Luciferase activity is then determined as described previously in step 3. Since compound is added only after the translation reaction is complete, inhibitors of translation should not score positively in this assay. Typically, a 1-ml in vitro translation reaction is sufficient to screen 45 candidate hits in duplicate for nonspecific luciferase inhibitory activity. Compounds that inhibit in this counterscreen are eliminated from future analysis. [Pg.320]

Proteins, blotted from polyacrylamide gels onto nitrocellulose sheets (Western blots) can be stained nonspecifically with a variety of dyes, or they can be identified individually by probing with appropriate antibodies. These procedures may be performed on duplicate blots, staining the total protein pattern on one blot and using the second blot for the immune reaction (1,2) This chapter describes how to combine both methods on one blot, i.e., staining the blot first for total protein, followed by an indirect immune reaction (3). [Pg.217]

Porcine anterior pituitary membranes were incubated with increasing concentrations (10 pM-900 pM) of [sHjn-propylapomorphine in the presence of 10 / M and 1 mM GTP and in the absence of GTP as the control. Nonspecific binding was determined in the presence of 1 mM (+)butaclamol and was the same with or without the addition of GTP. Data were analyzed as described in Figure 1. The number of sites labelled by [3H]n-propylapomorphine was 26.7 pM (absence), 19.6 pM in the presence of 10 / M GTP and 6.0 pM in the presence of 1 mM GTP and dissociation constants for the ligand were 280 and 210 pM. The experiment was performed in duplicate and is representative of three such experiments. [Pg.79]

Porcine anterior pituitary membranes were incubated as described in Figure 1 with [3HJspiro-peridol (10-1100 pM). Nonspecific binding was determined in the presence of 1 mM (- -)buta-clamol. Data were analyzed by curve fitting with a model for the binding of the radioligand to one or two classes of sites (14). Data are presented in the Scatchard plot coordinates. The experiment was performed in duplicate and is similar to several other experiments with the same results but it represents an extreme case of the ability of the [3H] antagonist to discriminate between the two forms of the receptor. The two dissociation constants calculated for the control curve are different by about 10-fold. (Reproduced with permission from Ref. 25. [Pg.81]

From the previous exercises, you should have assessed the dilution of test serum that can be used to discriminate between positive and negative nonspecific results, based on the difference noted between the selected positive and negative sera titrated over full-dilution ranges. We are now going to titrate all the sera at the dilution found as duplicates (two wells per serum dilution in the indirect ELISA). [Pg.183]

To estimate specific GABAA-receptor-mediated Cl flux, nonspecific C1 uptake determined in the presence of the GABAA-receptor blocker PTX should be subtracted from the C1 uptake assayed in the absence of the blocker It is essential that the specific and nonspecific Cl uptake be determined in tandem (i.e., alternating) to cucumvent time-dependent differences because of longer series Measurements should be earned out at least in duplicate. Only those Cl uptake values that amount to 100% or more above the background binding of C1 to the filters are to be taken into account for the uptake measurements... [Pg.56]

Count duplicate 10 jjL aliquots of the stopped suspensions (specific and nonspecific uptake and background) and calculate the total amount of chloride in each incubation, assuming that 10 pL represents 10/1080, or 0,93% of the total dpm. The amount of accumulated in the synaptoneurosomes is expressed in nmol Ch by comparison with the total amount of Cl" m the assay, and normalized to the amount of synaptoneurosomal protein (in mg)... [Pg.58]

Some studies have attempted direct injection of DNA into tumors, either alone or in complex with lipids. Introducing DNA or RNA directly into the tumor limits immune responses sometimes elicited when viral preparations are administered. However, the efficiency of lipid-DNA to enter cells in a tumor remains a problem, as does the relatively nonspecific cellular uptake of lipid-DNA complexes. Attempts to incorporate molecules in the lipid bilayer that bind to cell surface receptor to improve cell specificity have been investigated, but the differential effects seen in vitro are not generally duplicated in vivo. Along the same line and more recently, nanoparticles has also been considered as vector for delivering therapeutic genes. An added benefit with nanoparticles is that they can also be imaged (Nie et al. 2007). [Pg.315]

Basophilia that is removed from duplicate sections by treatment in ribonuclease solution before staining is attributed to RNA. Nonspecific extraction is detected in control sections stained after exposure to the solvent without enzymes. To minimize the extraction of RNA by the buffer alone, Pearse advises a digestion temperature no higher than 37°C. (50). The particular method used to color the RNA is not critical. As the use of ribonuclease is expensive and somewhat troublesome, various chemical extractives of doubtful specificity, e.g., acids, have been proposed as alternatives (106, 107). [Pg.637]

As the methods in use for the coloration of carbohydrates in tissue sections lack sufficient specificity, additional evidence is needed for the presumptive identification of particular polysaccharides and glycoproteins. Various enzymes have been applied to either tissue sections or fresh tissue for the purpose of either solubilizing or blocking particular substrates. To rule out nonspecific solution, duplicate control sections are exposed to the solvent without enzyme or to an enzyme solution that has been inactivated. Afterwards, both the digested and the control sections are stained by a suitable method and then compared. Sections known to contain the substrate should be tested along with the unknown. Material that is colored in the control section but absent after enzyme treatment is presumptively identified as a substrate of the enzyme used. [Pg.638]


See other pages where Nonspecific duplices is mentioned: [Pg.288]    [Pg.288]    [Pg.55]    [Pg.151]    [Pg.77]    [Pg.88]    [Pg.45]    [Pg.265]    [Pg.377]    [Pg.242]    [Pg.2224]    [Pg.1548]    [Pg.12]    [Pg.225]    [Pg.324]    [Pg.101]   


SEARCH



Duplication

Nonspecificity

© 2024 chempedia.info