Filter binding radioactivity assays

A related approach is realized in filter binding assays. Here the reaction solution is filtered, e.g., through nitrocellulose where proteins are absorbed, while small molecules can pass. One example of this technique is the quantification of protein bound and free nucleotides (with radioactive labeled ligands). 

The assay is performed by mixing trace amounts of radioactively labelled RNA with protein at different concentrations and then filtering through a nitrocellulose filter. Ideally, 100% of the RNA should be retained at high protein concentrations, but in reality a lower retention efficiency (percent RNA retained in the presence of a large excess of protein) is obtained. In addition, a fraction of the protein might not be retained by the filter. As an example, filter binding analysis of the 5 S rRNA-L18 complex revealed that the retention efficiency was 35% for the 5 S rRNA-L18 complex while 65% of the L18 protein was retained and <5% of free 5 S rRNA was retained.1 However, the accuracy of the measurements does not depend directly on the retention efficiency, provided a constant proportion of the complexes are retained at the concentrations tested. 

TCA precipitation may be replaced by DE81 filter-binding assay (20). Actually, this entire step may be omitted m routine expenments and replaced by simple counting of an aliquot of the final reaction product (resuspended e.g, in 100 xL). Note, however, that a fraction of the unincorporated radionucleotides may not be eliminated after the two ethanol-precipitation steps. Therefore, a final radioactivity count may overestimate the actual amount of probe. 

In 1987, CL started to be applied in DNA hybridization assays as an alternative to the use of radioactive tags. These assays are based on the specificity of a binding process that of DNA strands for each other. An unknown DNA can be identified with the Southern blot method in which the strands of the analyte are separated and allowed to interact with labeled probe DNA strands on nitrocellulose filter paper. If the label on the probe is detected, the DNA can be identified and, in some cases, quantitated. Conventionally, radioactive tags were used be-... 

Figure 2 Effect of pertussis toxin treatment on the regulation of basal [35S]GTP-yS binding by (2S,3R)TMT-L-Tic in hDOR/CHO cell membranes. Chinese hamster ovary cells stably expressing the human delta opioid receptors (hDOR/CHO) were treated in the presence ( ) or absence ( ) of 50 ng/mL pertussis toxin for 18 h. The cells were washed and cell membranes prepared as previously described [40]. Membranes were incubated with appropriate concentrations of (2S,3R)TMT-L-Tic in the presence of 0.1 nM [35S]GTPyS (1,250 Ci/mmol) in 1.0 mL of assay buffer (25 mM Tris, 150 mM NaCl, 50 iM GDP, 2.5 mM MgCl2, 1 mM EDTA, 30 pM bestatin, 10 pM captopril, pH = 7.4). After 90 min incubation at 30°C, the reaction was terminated by rapid filtration. The filters were washed with 25 mM Tris/120 mM NaCl, pH 7.4, and bound radioactivity was measured by liquid scintillation spectrophotometry.

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... 

Optimal Amounts of Reagents. The functional purity of [ I]PA and the optimal amounts of beads and tracer to use for routine assay of fluid-phase (PA reactive) IgG are determined from the binding curves shown in Fig. 1. Increasing amounts of beads (0.1 ml 5-200 /ag of beads corresponding to 15-600 ng of rabbit IgG) are incubated for 60 min at 30° with [125IJPA (0.1 ml). The beads are washed with two 3-ml portions of buffer by centrifugation at 1500 g (4°) for 5 min or by filtration on polycarbonate filters, and the radioactivity in the bead pellets is determined. In this ex-... 

C-labeled histidine-tRNA. Only the aminoacyl-tRNA whose binding is directed by the trinucleotide codon will become bound to the ribosomes and retained on the nitrocellulose filter. The amount of radioactivity retained by the filter is a measure of trinucleotide-directed binding of a particular labeled aminoacyl-tRNA by ribosomes. Use of this binding assay to test the 64 possible codon trinucleotides against the 20 different amino acids quickly enabled researchers to assign triplet code words to the individual amino acids. The genetic code was broken. 

In the binding assay technique, various tRNA molecules, one of which is radioactively labeled with are mixed with ribosomes and synthetic trinucleotides bound to a filter. If the radioactive label is detected on the filter, then it is known that the particular tRNA bound to that triplet. The binding experiments can be repeated until all the triplets are assigned. 

As for the binding assay if a radioactive ligand is available, try the filter assay first. It s the fastest and its applicability usually only depends on whether or not the ligand sticks to the filter. You should be able to answer this question after a few experiments. If the filter test does not work, use the column assay. If that is not possible (e.g., because ligand and binding protein are about equal in size), you can employ PEG precipitation or the methods from Petrenko et al. (1990) or Scheer and Meldolesi (1985). 

A variety of in vitro assays have been developed that minimize or avoid live animal experimentation. Several capitalize on the sodium channel s affinity for these toxins. Neuronal cell lines lyse in the presence of veratridine, a sodium channel activator, and ouabain, which prevents removal of the excessive sodium ions allowed in by veratridine. In the presence of both these drugs, a sodium channel blocker such as saxitoxin rescues the cells. Cellular viability can then be measured by adding tetrazolium salts that are metabolized by living cells to a colored product. Alternatively, isolated cellular membranes, typically from brain tissue, are used to bind radiolabeled saxitoxin. After incubating receptor and radioligand in the presence of a test sample, any radiolabeled saxitoxin bound to the cell membranes are deposited onto filters by vacuum pressure. Radioactivity from the labeled saxitoxin is then measured with a signal reduction indicating the presence of saxitoxin. 

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