Protein scintillation counter

Fluorescent materials are very important in medical research. Dyes such as fluorescein (21) can be attached to protein molecules, and the protein can be traced in a biological system by exciting the fluorescein and looking for its emissions. The use of a fluorescent material allows the detection of much smaller concentrations than would otherwise be possible. Because fluorescent materials can be activated by radioactivity, they are also used in scintillation counters to measure radiation (see Box 17.2). 

Concentrations of radiolabeled proteins, substrates, or products can be quantified by scintillation counters, which detect both emitters of weak (e. g., 3H) and high energy (e.g., 32P) by excitation of an organic solvent (e.g., toluene) which then emits fluorescence fight. In commercial systems the primary fluorescence is transformed via one or two additional fluorescent dyes in the solution into a visible emission signal which can easily be detected by conventional photomultipliers. 

Both the organic and water layers from the radioactive insecticides were counted in a liquid scintillation counter (Isocap/300, Searle). The results are expressed as nanomoles or micrograms equivalent of the substrate per mg protein. 

Add 200 pi water to the cell pellet and sonicate briefly at low power. Use 50 pi homogenate for a protein determination and 100 pi for lipid extraction. To 100 pi homogenate add 4 ml chloroform/methanol 2/1 (v/v). Leave for 1 h at room temperature and add 0.8 ml of 0.73% NaCl. Vortex and centrifuge for 5 min at 1000xg to separate the phases. Remove the lower chloroform phase and add 10 nmol cho-lesteryl oleate and 10 nmol triolein as a cold carrier. Dry the sample under nitrogen. Take the sample up in 25 pi chloroform/methanol 2/1 (v/v) and spot the sample on a silica gel plate (0.5 cm streak). Rinse the tube with 25 pi chloroform/methanol 2/1 (v/v) and spot the sample on a silica gel plate. Develop the plate with hexane/diethyl-ether/acetic acid, 70/30/1 (v/v/v). Remove the plate from the tank when the solvent front almost reaches the top, dry the plate, and expose briefly to iodine vapor to localize the cholesteryl oleate. Scrape the spots into scintillation vials, mix thoroughly, and count in a scintillation counter. 

Radioactive decay with emission of particles is a random process. It is impossible to predict with certainty when a radioactive event will occur. Therefore, a series of measurements made on a radioactive sample will result in a series of different count rates, but they will be centered around an average or mean value of counts per minute. Table 1.1 contains such a series of count rates obtained with a scintillation counter on a single radioactive sample. A similar table could be prepared for other biochemical measurements, including the rate of an enzyme-catalyzed reaction or the protein concentration of a solution as determined by the Bradford method. The arithmetic average or mean of the numbers is calculated by totaling all the experimental values observed for a sample (the counting rates, the velocity of the reaction, or protein concentration) and dividing the total by the number of times the measurement was made. The mean is defined by Equation 1.1. 

Information concerning the detailed structural features of the protein is contained in the intensities of the diffraction spots. All the atoms in the protein structure make individual contributions to the intensity of each diffraction spot. Therefore, to deduce the three-dimensional structure, all the spots must be measured, either by scanning the x-ray films with a densitometer or by measuring the diffraction spots individually with a scintillation counter. 

An in vitro reticulocyte translation assay (Boehringer Mannheim) was modified to determine an inhibition of translation by alkaloids. An assay (total volume 25 jl) contained 2 jl 12.5 x translation mix (Boehringer), 10 pi reticulocyte lysate, 200 mM K-acetate, 1.5 mM Mg-acetate, 0.25 pCi L-[4,5-3H(N)]-leucine, 0.5 pg TMV-RNA (Boehringer) and up to 5 mM alkaloids (buffered to pH 7). The mixture was incubated at 30 °C reactions were terminated after 0, 10, 20, 30 and 40 min. The radiolabeled protein was precipitated by adding 200 pi ice-cold trichloroacetic acid (TCA) (50% w/v) and, after 30 min, filtered through GF 34 filters (Schleicher-Schull), which binds proteins. After washing the filters three times with 50% TCA, they were dried at 85 °C. Radioactivity of the filters was determined in a liquid scintillation counter.19... 

Muscarinic receptor (mAChR). Membrane preparations adjusted to 500 pg protein in a final volume of 500 pL buffer were incubated with [3H]-quinuclidinyl benzilate (QNB) (52.3 Ci/mmol Dupont NEN) for 1 h at 20° in the absence and presence of alkaloids, employing 20 pM atropine as a positive control substance. The incubation was stopped with 3 mL ice-cold 0.9% NaCI-solution and filtered (by suction) through Whatman GF/C glass fiber filters. The filters were washed three times with 3 ml 0.9% NaCI, placed in vials, and dried for 30 min at 60°C. Their radioactivity was measured in a liquid scintillation counter (RackBeta, Pharmacia) using Ultima-Gold" (Packard) as the scintillation cocktail. 

Exactly 1.7 mg of a purified enzyme (MW =55,000) was incubated with an excess of iodoacetamide-C (S.A. = 2 fiCii mmole). The car boxy methylated protein was then precipitated, washed free of unreacted iodoacetamide-C , dissolved in a small amount of buffer, and the entire solution counted in a scintillation counter operating at 80% efficiency. In one hour, the sample gave 13,190 counts above background. Calculate the number of reactive SH groups per molecule of protein. 

Measure incorporated or 14C-protein hydrolysate in a b-scintillation counter. Plot the concentration against the 14C- uptake values and calculate the 50% cytotoxicity value (CC50) (Table 1). 

When a similar fat load, was used without the inclusion of protein and carbohydrates, peak C02 occurred later in some patients. In 53 patients in whom CO2 excretion peaked within 6 hours, the sensitivity and specificity of the tests were 100% and 96%, respectively. Breath samples for C02 measurement are not easily transportable, and the requirement for a scintillation counter and the administrative regulations surrounding the use of radioisotopes deter many laboratories from using the test. 

If film is used as detector this control is, of course, not available. Instead an absorption edge scan needs to be made. Since the metal concentration in the protein sample is very low, it is necessary to use several scintillation counters aimed at the crystal sample to pick up the crystal sample fluorescence. 

Protein synthesis was determined in the non-infected cells used as control and the HSV-1 and/or VSV infected cells, in order to obtain quantitative data on the toxicity and antiviral activity of the compounds assayed. Thus, 0.5 free medium methionine and 1 pCi/ml of methinione 32S were added to die cells and incubated at 37°C for 1 h. Thereafter the medium was siphoned off and the cells were washed with saline phosphate buffer and precipitated with 5% trichloroacetic acid (TCA). After 5 min, the acid was eliminated and the cells were washed twice with 96% ethanol,dried under an infrared light and dissolved in NaOH 0.1N with DSS. A liquid scintillation counter was used for the recount. The results showed no antiviral activity for die dihydro-f -agarofuran sesquiterpenes. 

The 50% inhibition concentration of erbstatin against tyrosine kinase was 0.55 p,g/ml, when it was examined as follows The reaction mixture contained 1 mM MnCl2, 100 ng EGF, 40 lg protein of A431 membrane fraction, 75 lg of albumin, 3 lg of histone, and HEPES (N-2-hydroxyethylpiperazine-N -2-ethanesulfonic acid) buffer (20 mM, pH 7.4) in a final volume of 50 (11. The reaction tubes were placed on ice and incubated for 10 minutes in the presence or absence of erbstatin. The reaction was initiated by the addition of labeled ATP (10 (11), and the incubation was continued for 30 minutes at 0°C. Then aliquots of 50 (11 were pipetted onto Whatman 3MM filter paper and put immediately into a beaker of cold 10% TCA containing 0.01 M sodium pyrophosphate. The filter papers were washed extensively with TCA solution containing 0.01 M sodium pyrophosphate at room temperature, extracted with ethanol and ether, and then dried. Radioactivity was measured by a scintillation counter. 

Cells were solubilized in 0.5 mL of 1% SDS, and radioactivity was determined using a Beckman Instruments (Palo Alto, CA) LS 6000 liquid scintillation counter at approx 50% efficiency. Cells from parallel wells were solubilized in 0.5 mL of 1N NaOH for protein amount measurements using a Bio-Rad Protein Assay solution (Bio-Rad) (see Fig. 4 and Note 1). 

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