Hybridization of polyuridylic acid to human DNA immobilized onto nitrocellulose filters.

The level of deoxyadenylate (da) regions in human DNA was estimated from formation of poly(U)-poly(da) triplexes on nitrocellulose filters that were RNAase resistant. The (dA) rich sequences were determined following mild ribonuclease treatment of the poly(U)-DNA hybrids (5 mug/ml at 25 degreesC for 30 min), where as exhaustive ribonuclease treatment (5 mug/ml at 25 degrees C for 6 hr) estimated the more (dA) pure sequences. The level of (dA) rich regions was 0.39% of the DNA and for the more (dA) pure regions it was 0.07%. The (dA) regions were widely distributed throughout human DNA regardless of base composition or sequence repetition. However, a concentration of (dA) regions into main band CsC1 gradient fractions of DNA and into repeated DNA was observed.     

Routine detection of calcium-binding proteins following their adsorption to nitrocellulose membrane filters

A routine semiquantitative procedure which permits soluble calcium-binding proteins to be detected following their adsorption to nitrocellulose membrane filters by liquid scintillation counting of specifically bound 45Ca is described. Proteins with high affinity for calcium such as calmodulin and troponin can be detected with a detection threshold of about 2 micrograms per 400 microliter. Modifications to decrease this limit are feasible and are discussed. This technique should allow calcium-binding proteins of unknown function to be assayed during their purification. It was necessary to treat solutions containing 45Ca with chelex-100 in order to prevent loss of calcium binding which occurred as the decay product (Sc3+) accumulated, suggesting that all studies utilizing 45Ca as a tracer should evaluate possible interference by this ion.     

Permeability and structure of ethanol-sterilized nitrocellulose (Millipore) filters

Ethanol sterilization of nitrocellulose (Millipore) filters did not alter the permeability for ³H-thymidine or bovine serum albumin, neither was the porosity changed. Contrary to a previous report ethanol pretreatment did not decrease the pore size judged from scanning electron micrographs. This was also confirmed by freeze-etching electron microscopy. Thus sterilization of nitrocellulose filters with 70 % ethanol seems to be a safe procedure for their use, e.g. in embryological and immunological experiments.     

Hydroxyapatite-mediated transfer of DNA on nitrocellulose filters in dot-hybridization experiments

The method of hydroxylapatite-mediated rapid and effective transfer of DNA onto nitrocellulose filters for following dot-hybridization was elaborated. The analysed DNA occurred initially in diluted and large volume solutions (from 1 to 10 ml) with various composition (2 M NaCl; 4 M LiCl--8 M urea; 4 M CsCl; 5 and 20% sucrose) was adsorbed on hydroxylapatite and quantitatively transferred onto nitrocellulose after hydroxylapatite solubilization in a small volume of acid (usually, 200 microliters of 10% TCA). As exemplified by the hybridization of total rat liver DNA with the plasmid ph22 DNA containing a cluster of sea urchin histone genes, the method presented appears to be not only simple and useful for handling multiple probes of diluted DNA solutions with high concentrations of salts, sucrose and urea but also more sensitive than some convenient DNA dot-hybridization methods.     

The binding of gyrase to DNA: analysis by retention by nitrocellulose filters.

Three distinct Escherichia coli DNA gyrase complexes with DNA can be identified using a nitrocellulose filter-binding assay. One complex consists of an ensemble of two subunit A and two subunit B protomers bound noncovalently to specific sequences of DNA. High levels of each subunit alone are inactive but a single gyrase molecule binds DNA to a filter. At 23 degrees, the complex has a dissociation constant of approximately 10(-10) M and a half-time of decay of about 60 h. It is sufficiently stable that it can be purified by gel filtration and retain full supercoiling activity. Gyrase binds preferentially to relaxed DNA over supercoiled DNA by a factor of about 10. On addition of oxolinic acid, a second complex is formed that is distinguished by its stability in high ionic strength solutions and by efficient conversion to a third form upon addition of protein denaturants. The first and second complexes require Mg++ for optimal formation. The third form has been shown previously to contain denatured A protomers covalently linked to DNA that is broken at the site of attachment.     

A simple assay of solubilized adenosine receptors with nitrocellulose membrane filters

A rapid and simple assay of solubilized adenosine receptors with nitrocellulose membrane filters is described. This assay was sensitive and reproducible when applied to adenosine receptors solubilized from rat brainstem membranes with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Appropriate values of dissociation constants for the solubilized adenosine receptors to their tritiated agonists were obtained by the membrane filter technique. This method should be applicable for the assay of a variety of solubilized receptors.     

Use of nitrocellulose Synpor filters for counting soil bacteria by epifluorescence microscopy

A modified acridine orange staining method for estimating soil bacterial numbers by epifluorescence microscopy using Synpor filters (VCHZ Synthesia, Czechoslovakia) was elaborated. Comparing with the method of direct count of soil bacteria estimated in a Bürker chamber higher counts of bacteria and a lower variation of results were obtained. To verify the sensitivity of the method, microflora from various soil horizons was tested.     

Visualization of acid phosphatase activity on nitrocellulose filters following electroblotting of polyacrylamide gels

A method for visualizing acid phosphatase isoenzymes by activity staining on nitrocellulose filters after electroblotting of proteins fractionated on nondenaturing polyacrylamide gels is described. Reproducible results were obtained when 25 mM Tris-192 mM glycine was used as the transfer buffer instead of 0.7% acetic acid, 50 mM sodium acetate, pH 4, or 0.14 M acetic acid--0.35 M beta-alanine, pH 4.3. Dot-blot analysis of banana fruit extracts on nitrocellulose filters revealed that a minimum of 5 x 10(-3) units (nmol p-nitrophenyl phosphate hydrolyzed g-1.h-1) of acid phosphatase activity can be detected. This method can be suitable for screening a large number of biological samples for monitoring acid phosphatase activity.     

A solid-phase assay for the phosphorylation of proteins blotted on nitrocellulose membrane filters

A new procedure for the phosphorylation and assay of phosphoproteins is described. Proteins are solubilized from tissue samples, separated by polyacrylamide gel electrophoresis, transferred onto nitrocellulose membrane filters, and the blotted polypeptides are phosphorylated with the catalytic subunit of cyclic AMP (adenosine 3':5'-monophosphate)-dependent protein kinase. The method was developed for the assay of dephosphosynapsin I, but it has also proven suitable for the phosphorylation of other proteins. The patterns of phosphorylation of tissue samples phosphorylated using the new method are similar to those obtained using the conventional test tube assay. Once phosphorylated, the adsorbed proteins can be digested with proteases and subjected to phosphopeptide mapping. The phosphorylated blotted proteins can also be analyzed by overlay techniques for the immunological detection of polypeptides.     

Comparisons using 35S- and 32P-labeled DNA for hybridization on nitrocellulose filters

DNA was labeled by nick translation with 35S and used as a probe in Southern- or colony-blot DNA hybridization. Comparison with DNA labeled with 32P showed that not only was 35S-labeled DNA suitable as a probe, but in many cases had advantages. The longer half-life of 35S allows for less stringent timing of experiments and eliminates the waste of unused old label. Resolution on autoradiographs was found to improve when using 35S-labeled DNA.     

Comparison of fluorographic methods for detecting radioactivity in polyacrylamide gels or on nitrocellulose filters

The commercial fluorographic enhancers, En3Hance or Amplify, were not as efficient as 2,5-diphenyloxazole (PPO) for detecting radioactively labeled proteins in polyacrylamide gels or on nitrocellulose filters. For most of the X-ray films tested, optimal preexposure was essential to obtain maximum sensitivity in fluorography or indirect autoradiography using intensifying screens. The best results were obtained with nitrocellulose by saturating the filters with PPO. The minimum levels of 35S/14C that could be detected on filters by autoradiography or fluorography in a 24-h exposure were 4 X 10(2) or 1 X 10(2) dpm cm-2 respectively. For 3H these levels were, respectively, 20 X 10(3) or 0.5 X 10(3) dpm cm-2.     

A new method of DNA and RNA transfer onto nitrocellulose filters during blot hybridization

A quick (1-2 hour) method of DNA and RNA transfer onto nitrocellulose filters for subsequent blot-hybridization was elaborated. The main features of the method proposed are, firstly, almost complete exclusion of the mechanical impact on the gel and, secondly, addition to the transfer medium (20 X SSC) of a chaotropic agent, 0.5 M NaClO4. The latter results in a slight dissolution of the gel matrix and, on the other hand, somewhat increases the binding of the nucleic acid to the nitrocellulose. The method shortens significantly the time of DNA or RNA transfer at equal, or even higher, quality of hybridization.