Lysates, measuring

Pyrogenic A fever-producing substance. The presence of these substances is determined by the Limulus Amebocyte Lysate (LAL) test and measured in EU/ml (endotoxin units per milliliter). 

An example of miR-dependent deadenylation of mRNA measured by this method is shown in Fig. 6.3C. In this case, HeLa cells (which express let-7) were transfected with a pDNA R-luc construct encoding three let-7 binding sites in the 3 UTR (3 X bulge), or with control constructs encoding either no sites (plasmid) or three mutated let-7 sites (3 x bulge mut) (constructs described in Pillai et al, 2005). Cells were harvested 24 h after transfection, RNA was purified for the PAT assay, and luciferase activity was measured from cell lysates. As reported previously, the presence of functional let-7 target sites results in specific repression of luciferase expression with very minor effects on mRNA stability (Pillai et al., 2005). The experiment in Fig. 6.3C demonstrates that the let-7 targeted reporter mRNA is selectively deadenylated. 

Cells are maintained in suitable medium and starved of serum, if required. Agonist/inhibitors are added for 30 min first, then [35S]Met (5 iCi/ml medium) is added for 60 to 120 min. Cells are then lysed and cleared lysates (equal amounts of proteins) are loaded onto 3 MM papers. After the samples have been allowed to soak into the paper, the papers are boiled in 5% (w/v) TCA three times (2 min each time), rinsed with 100% ethanol, and oven dried. [35S]methionine incorporation is measured in 3 ml of scintillation fluid. 

Hipp S, Walch A, Schuster T, et al. Precise measurement of the E-cadherin repressor Snail in formalin-fixed endometrial carcinoma using protein lysate microarrays. Clin. Exp. Metastasis 2008 25 679-683. 

The apparent retinal influx clearance,. Kin,retina expressed as mL/(min g retina), of the test substrate labeled with either [3 H] or [14C] from the circulating blood to the retina is determined by integration plot analysis. In brief, rats are anesthetized, followed by injection of the test substrate (e.g., an [3H]-labeled compound, about 10 /u.Ci/head) into the femoral vein. After collection of plasma samples, rats are decapitated and the retinas removed. The retinas are dissolved in 2 N NaOH and subsequently neutralized with 2 N HC1. The radioactivity of retinal cell lysates is measured by liquid scintillation spectrometry. As an index of the retinal distribution characteristics of the radiolabeled test substrate, the apparent retina-to-plasma concentration ratio (Vd) as a function of time is used. This ratio [Vd(Q] (mL/g retina) is defined as the amount of [3H] per gram retina divided by that per milliliter plasma, calculated over the time-period of the experiment. The Kjn,retina can be described by the following relationship ... 

Determination of nucleic acid yield. Cleared lysate or ImuVert was diluted to a concentration which had an absorbance between 0.4 and 0.5 at 260 nm and the absorbances at 280 and 260 nm were measured. The nucleic acid concentration in solution was then calculated by the method of Warburg and Christian (18), The yield of nucleic acid was calculated by determining the percent of nucleic acid in the lysate that was isolated in the product. The acceptable range for ImuVert manufacturing is 10.5 + 1.5. 

EMSA assays can also be exploited to measure STAT nuclear localization, which is, similar to NFkB localization, a measure of STAT activity. Determination of JAK phosphorylation is carried out by immunoprecipitation of the JAK proteins from cell lysates, followed by SDS-PAGE electrophoresis, immunoblotting with antiphosphotyrosine antibody and JAK-specific antibody re-probing [99]. 

The ELISA of cellular DNA fragmentation is a sandwich ELISA that measures apoptosis by quantitating the fragmentation and/or release of BrdU-labeled DNA. The commercial kit (Roche) can detect DNA fragments in the cell-free supernatants from cultured cells or cytoplasmic lysate of apoptotic cells prelabeled with BrdU... 

ELISA has been used for measuring caspase activity. For the ELISA of intracellular caspase activity at the very early stages of apoptosis, apoptotic cells are first lysed to isolate their intracellular contents. Different caspase activities in the cell lysate can then be determined by the addition of a caspase-specific tetrapeptide substrate that is conjugated to the color reporter molecule p-nitroanilide (pNA) (e.g., DEVD-pNA for caspase-3 and lETD-pNA for caspase-8). The cleavage of the substrate peptide by the caspase releases the chromophore pNA, which can be... 

Fig. 12. Effect of spontaneous apoptosis on the activation of caspase-3 in human eosinophils. Eosinophils (2 x 10 /ml) were cultured for 3 h. Cells were lysed and caspase-3 activity was measured by caspase-3 colorimetric assay kit (R D Systems). Enzymatic products were measured at 405 nm with BIOTEK EL340 ELISA microplate reader (BIO-TEK Instrument Inc., Vermont). Human recombinant caspase-3 (5 U) (Calbiochem, California) was used as a positive control. The stimulation index was determined by direct comparison to the level of the normal control. Background readings from cell lysates and buffers were substracted from the readings of both induced and uninduced samples samples before calculating the stimulation index in caspase-3 activity. The differences between control and treated groups were assessed by Student s i-test. P < 0.05 P < 0.001 (Z2).

In this study, a machine learning model system was developed to classify cell line chemosensitivity exclusively based on proteomic profiling. Using reverse-phase protein lysate microarrays, protein expression levels were measured by 52 antibodies in a panel of 60 human cancer cell (NCI-60) lines. The model system combined several well-known algorithms, including Random forests, Relief, and the nearest neighbor methods, to construct the protein expression-based chemosensitivity classifiers. 

Besides the enzymatic incubation in the reaction mixture, all procedures are carried out at 4°C. GTPCH activity is assayed by measuring the neopterin produced upon enzymatic incubation at 37°C for 60 min in a final volume of 0.1 ml in the dark (due to light sensitivity of pterins), followed by chemical oxidation and dephosphorylation. Two separate blanks are prepared, a blank reaction with cell lysate that is immediately oxidized to detect the neopterin that was present in the lysate, and a blank reaction without cell lysate to detect the neopterin that is generated from the incubation (substrate) buffer. The sum of both blanks is later subtracted from the value of the incubation reaction to determine the enzymatically produced neopterin. 

DHPR activity is assayed by measuring the linear increase of ferrocytochrome c at 550 nm (AEssonm = 21) upon enzymatic incubation at 25°C for 5 min in a volume of 1 ml. The incubation mixture contains 40 tl lysate, 875 tl of 50 mM Tris-HCl pH 7.6, 50 pi of the 1 mM ferricytochrome solution, and 25 pi of the 2 mM NADH solution. The blank control contains 40 pi of Tris-HCl pH 7.6 instead of the lysate. These mixtures are preincubated for 5 min at 25°C, before the reaction is started by addition of 10 pi of the 1 mM 6-MPH4 substrate. The reaction is monitored by measuring the AE550nm/min over a period of 5 min. The starting lysate of 40 pi was diluted 25-fold. 

Babior, who has studied this enzyme at several stages of its purification, found in lysates of PMNs which were activated with zymosan that of eight potential biological reductants only reduced pyridine nucleotides supported the formation of O ". The K , for NADPH was less than the K , for NADH and the activity was decreased in preparations from three patients with chronic granulomatous disease. In accord with predictions based on reaction 7, 0.55 molecule of O7 was measured per molecule of NADPH oxidized under conditions of saturating concentrations of cytochrome c The enzyme which was extracted with Triton X-100 from a granule-rich fraction from activated PMNs, required an external source of FAD for the formation of O from NADPH . Riboflavin and FMN would not substitute. Flavin adenine dinucleotide was proposed as a necessary cofactor, which was probably lost when the enzyme was treated with the detergent. 

Fig. 5.1 Principle behind the CALUX bioassay for Ah-receptor agonists. Following binding of the agonist to the Ah-receptor, the complex will be transported to the nucleus and bind to a so-called dioxin responsive element, resulting in the increased transcription of the luciferase gene and production of luciferase. Following incubation this enzyme can subsequently be measured in cell lysates by a light producing reaction.

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