Filter-binding assay

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. 

A number of parameters influence the retention efficiency of the assay and should be optimized for each binding protein studied Buffer Increasing salt concentrations lower the retention efficiency. For most complexes the monovalent ion concentrations should be above 150 mM to ensure specificity of binding. 

Temperature Low temperature increases the retention efficiency. Flow rate and washing conditions The flow rate should be kept constant, i.e. apply the same suction in each experiment. Unspecific binding of naked RNA to the filter can be reduced by extensive washing, but often at the expense of the retention efficiency. It is for some complexes an advantage with consecutive (3-4) washes with small volumes, while for other complexes the best results are obtained when the complex is applied in a larger volume. 

RNA renaturation It is well known that different RNA preparations give variable retention efficiencies. Careful renaturation often reduces the variability between different preparations (see Section 2.1.2.6). 

Soak and degas the nitrocellulose filter in the binding buffer Binding buffera 30 mM Tris-HCl, pH 7.8 10 mM MgCl2 150 mM KC1 

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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). 

As described for equilibria before, separation techniques like filter binding assays or pull down methods can be applied to separate educts and products of slow reactions too. Here modern HPLC systems with autosampler and time programmed injection offer a convenient approach for the analysis of reactions in the time scale of minutes and hours. 

GTP hydrolysis by Ras, which is considerably slow, has been analyzed, usually by single turn-over measurements. Ras is loaded with GTP by incubation of Ras (being in the GDP form after preparation) with a large excess of GTP in the presence of EDTA, which leads to faster dissociation of the nucleotide. After change of buffer and removal of excess nucleotide, aliquots are taken at certain time intervals and the nucleotide composition is analyzed with the help of filter binding assays or HPLC. Series of experiments with mutations in the neighbor-... 

The first two rounds are performed under low stringency conditions to enhance RNA-protein binding and to avoid early depletion of sequences present in the SELEX RNA pool. For SELEX cycles 1-3 a nitrocellulose-filter binding assay is used to separate receptor-bound from free aptamers. Beginning from SELEX cycle 4, the nitrocellulose-filter binding and a gel-shifr selection step are employed as two consecutive selection processes (see Note 2). 

Haugan IR, Nilsen BM, Worland S, Olsen L, Helland DE. Characterization of the DNA-binding activity of HIV-1 integrase using a filter binding assay. Biochem Biophys Res Commun 1995 217 802-810. 

The modified cellulose filter binding assay is based on the tight binding of proteins to this kind of filter material. When a protein-nucleic acid mixture is filtered, proteins are retained on the filter while nucleic acids are washed through. However, nucleic acids are also retained on the filter if they are bound to proteins. Thus, free and protein-bound nucleic acids can be separated [43]. 

Other filter binding assays, like the nitrocellulose binding assay, have been used very successfully to investigate the interaction of proteins with various nucleic acids (Riggs et al. 1970). This method does not rely on differences in size to separate free and bound species, but on the fact that proteins are selectively bound to the mem-... 

Because of the nature of the chip surface, BIA is a heterophasic technique like the GEMSA and filter binding assays discussed in the previous section. There have been careful analyses made of the potential effect of artefacts arising from the heterophasic nature of the chip, such as steric hindrance and mass transport effects, on the absolute values of rate and thermodynamic parameters (Schuck 1997). The conclusion appears to be that relative values of parameters, particularly equilibrium constants, obtained by this approach can be viewed with confidence, but careful experimental design is needed to determine reliable absolute values (Myska 2000). 

In another approach, a filter-binding assay was found to be of value to study the kinetics of triplex formation by 19-mers in the R RY motif [40]. It was found that the Kojf is pH dependent owing to a rapid acid-base equilibrium of... 

The individual sequences can be tested for their affinity for the target protein by various methods like filter binding assay, gel-shift, and surface plasmon resonance. The individual sequences from each class can be synthetically prepared from DNA synthesis facilities and tested for their ligand-binding capability. A representative binding curve from which the binding affinity can be estimated is shown in Fig. 3. 

Using a membrane filter binding assay, the competition between poly(ADP-ribose) and DNA for binding of individual histones was measiued (3). The size of the poly(ADP-ribose) molecules firom rat liver nuclei ranged from 10 to 60 monomer units (3). Fig. 1 shows the effect of unlabeled... 

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