Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Polyacrylamides”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 883 records · Page 49Linked to original sources
PubMed1996

Development of a quantitative polyacrylamide gel electrophoresis analysis using a multichannel radioactivity counter for the evaluation of oligonucleotide-bound drug carrier.

A quantitative polyacrylamide gel electrophoresis (PAGE) analysis using a multichannel radioactivity counter was designed for the evaluation of 33P-labeled antisense oligonucleotide associated with polymeric drug carrier (nanoparticles). The proposed analytical method was first validated. The criteria of specificity, linearity, reliability, detection and quantification limits, and resolution power were determined. Results were compared to those obtained using liquid scintillation counting of crude samples or after solubilization of gel slices. The proposed method gave a better linearity and reliability than liquid scintillation counting of solubilized gel slices. In comparison with the liquid scintillation counting of crude samples, the method presented the advantage of being able to directly separate oligonucleotides differing by only one nucleotide in length. This method was applied for the separation of free oligonucleotides and oligonucleotides bound onto nanoparticles, allowing quantification of the amount of free and bound oligonucleotides without any further separation steps. Thus, because it is easy and rapid, the quantitative PAGE analysis using a multichannel radioactivity counter offers interesting possibilities for the characterization of oligonucleotide nanoparticles.

Cyanoacrylates↗
PubMed1996

Rapid electrophoretic analysis of heterogeneity of bovine serum albumin using a linear polyacrylamide-coated capillary.

Bovine serum albumin was separated into three major peaks and two or three minor peaks at pH 7.4 within 12 min by capillary zone electrophoresis using a linear polyacrylamide-coated capillary. The two major peaks, having medium and low mobilities, were assigned to the monomers of nonmercaptoalbumin and mercaptoalbumin, respectively. On the other hand, cross-linked albumins were distributed as a broad peak around the major peak with high mobility. Thus, commercially available bovine serum albumins from various sources were revealed to have a wide variety of heterogeneity in terms of their respective components.

Animals↗
PubMed1996

Negative staining of proteins in polyacrylamide gels with methyl trichloroacetate.

This paper describes a new, sensitive (in the nanogram range), and rapid (two-step) technique for the negative staining of proteins in polyacrylamide gels in the presence or absence of sodium dodecyl sulfate. After separation, gels are incubated with 8% methyl trichloroacetate ester in 38% isopropanol and then washed in water to produce a negative image of colorless proteins against an opaque background. The technique allows unmodified proteins to be recovered for biological studies or transblot for amino acid sequence. Finally, owing to the reversibility of the process, gels can be restained after rapid visualization. For these reasons, negative staining with methyl trichloroacetate should become the method of choice for rapid and sensitive staining of proteins prior to further processing, including stable staining with silver ions.

Chemical Precipitation↗
PubMed1997

Polyacrylamide gel electrophoresis in discontinuous transverse urea-gradient gels.

Polyacrylamide gel electrophoresis in transverse urea-gradient gels is widely used for the study of changes in conformation and quaternary structure of proteins induced by increasing concentrations of urea. However, possible uncertainties regarding the effective concentration of urea at any given point across the gel must be considered when this technique is used for quantitative purposes. We describe here a simple, practical procedure for preparing urea-gradient gels discontinuously, by stacking various layers of acrylamide solution with increasing urea concentrations. We show that the urea concentration in different lanes of discontinuous gradient gels prepared in this way can be predetermined accurately enough for quantitative purposes and provide as an example the study of the dissociation of thyroglobulin in urea. Various levels of resolution can be attained by this technique. The use of discontinuous gradient gels may extend the usefulness of urea gel electrophoresis in relation with the quantitative aspects of the study of protein conformation.

Electrophoresis, Polyacrylamide Gel↗
PubMed1997

Analysis of single-strand conformation polymorphism by capillary electrophoresis with laser-induced fluorescence detection using short-chain polyacrylamide as sieving medium.

A rapid analysis of single-strand conformation polymorphism (SSCP) by capillary electrophoresis with laser-induced fluorescence (CE-LIF) detector was developed using a short-chain, linear polyacrylamide (PA) as sieving medium. Capillary filling of this low-viscosity medium and medium replacement were carried out by commercial capillary electrophoresis instruments. The approach was successfully applied to detect the C677T mutation of methylenetetrahydrofolate reductase gene. The influences of factors such as the concentration of polymers, voltage, temperature, and additives on the SSCP analysis were systematically investigated. Using 6% PA sieving medium and high electric field, four strands were resolved within 11 min in a DNA sample heterozygous for the C677T mutation, and a characteristic pattern was apparent for each of the three genotypes. When using multiple injection mode, the average analysis time per sample was reduced to about 4 min. In conclusion, our results indicate that CE-LIF may be an alternative to conventional SSCP analysis based on slab gel electrophoresis for the detection of genetic mutations. The technique is simple and rapid and is well suited to analysis of large numbers of clinical samples.

Acrylic Resins↗
PubMed1997

High-efficiency blotting of proteins of diverse sizes following sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

High-molecular-weight proteins often blot onto nitrocellulose membranes poorly following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, resulting in low levels of detection on immunoblots, and hence difficulty in analyzing rare proteins. Moreover, optimizing conditions for the transfer of high-molecular-weight proteins to nitrocellulose frequently results in the inefficient transfer or loss of lower molecular weight proteins. This problem is particularly vexing during the analysis of large proteins which may be processed to one or more smaller biologically active forms. Using radiolabeled protein standards and phosphorimaging technology, we have quantitated the efficacy of many different protein gel electrophoresis and blotting protocols. Here we report novel gel and blotting conditions which significantly improve the transfer and retention of high-molecular-weight proteins, without sacrificing the efficient transfer of lower molecular weight proteins. Using this newly described procedure, we have detected a rare 500-kDa protein in immunoblots which was previously not detectable with any of the commonly used blotting procedures. Since the improved conditions offer increased sensitivity across a spectrum of protein sizes, they should be widely applicable.

Blotting, Western↗
PubMed1997

Micropreparative high resolution purification of proteins by a combination of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing, and membrane blotting.

We report a simple, economical, and efficient protocol for protein purification from cells. First, proteins of cell lysates were separated by standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted to protein-blotting membrane. The blots were stained with Coomassie blue or developed by immunoblotting to visualize specific proteins. The bands corresponding to those visible by immunoblotting were excised from the dye-stained blots and subjected to isoelectric focusing. The focused gel was stained with Coomassie blue. Finally, the stained bands were excised and subjected to another SDS-PAGE separation and electrotransferred back to protein-blotting membrane. At this stage, the purified proteins were suitable for microsequencing. We have tested the feasibility of this novel technique by purifying proteins with molecular weights ranging from 19 to 100 kDa from a lysate of Sarcocystis neurona, the etiologic agent of equine protozoal myeloencephalitis. The purity of proteins was demonstrated by reverse-phase high-performance liquid chromatography. Partial sequences of these purified proteins were obtained by N-terminal or digestive sequencing.

Animals↗
PubMed1997

The staining of acidic proteins on polyacrylamide gels: enhanced sensitivity and stability of "Stains-all" staining in combination with silver nitrate.

A number of acidic proteins, such as those found in bone and dentin, are poorly resolved on acrylamide gels using Coomassie blue or silver nitrate staining. The cationic dye Stains-all allows visualization and identification of these proteins due to their differential staining: highly acidic proteins stain blue and intact proteoglycans stain purple, whereas less acidic proteins stain pink. However, the use of Stains-all is limited due to relatively poor staining sensitivity and lack of stability to light. A procedure which addresses these deficiencies has been developed utilizing established protocols for Stains-all staining followed by silver nitrate incubation and development. In this way, phosphoproteins such as osteopontin, bone sialoprotein, dentin phosphophoryn, and other acidic glycoproteins are visualized at higher sensitivity (greater than fivefold) and staining stability than normally achieved with just Stains-all. The protocol stains a greater variety of proteins than a combined alcian blue/silver staining procedure previously described. Utilizing the Stains-all/silver protocol, porcine bone osteopontin, a protein not visualized by standard silver staining, can be observed in amounts as little as 0.25 ng on polyacrylamide gels. Furthermore, densitometric scans demonstrate that the staining intensity is proportional to osteopontin amount and can be used for quantification over a range from 0.25 to 50 ng.

Acrylic Resins↗
PubMed1997

Semidry electroblotting of peptides and proteins from acid-urea polyacrylamide gels.

A semidry electrophoretic transfer method was developed for efficient electroblotting of proteins separated by acid-urea polyacrylamide gel electrophoresis (AU-PAGE). Model polypeptides ranging from 1.8 to 21.5 kDa were used to test transfer parameters that included time of transfer, power settings, transfer solutions, and membrane type. Optimized conditions were identified which allowed for transfer efficiencies of 70-100% following 5-15 min of applied current. The best transfer solution was 5% acetic acid, the same solvent used for electrophoresis. Therefore, acid-urea gels could be subjected to electrophoretic transfer without a soaking step, thereby reducing loss of band resolution and eliminating leaching of protein from the gel. The method was shown to be applicable to Western blot analysis of rat neutrophil defensins.

Acetic Acid↗
PubMed1998

Elution of lipopolysaccharides from polyacrylamide gels.

A simple procedure for elution in water of bacterial lipopolysaccharides (LPS) from sodium dodecyl sulfate-polyacrylamide gels is described. It consists of the combination of three principal steps: first, highly sensitive on-gel LPS detection (1-10 ng/band) with zinc-imidazole (negative or reverse staining); second, washing of the individual LPS band in a solution of a zinc-complexing agent (e.g., 100 mM EDTA); and finally, elution of the LPS (100-200 microliters water for a 0.5-microgram LPS band) from gel microparticles for 3 h at room temperature. Using this procedure, we have successfully eluted a variety of LPS forms from Bordetella pertussis, Escherichia coli 0111:B4, E. coli K-235, Salmonella enteritidis, and Pseudomonas aeruginosa. Elution recovery of rough or semismooth LPS was about 70-80%, while that of smooth LPS was only about 10%. Eluted LPS was biologically active as tested by limulus amebocyte lysate and TNF-alpha assays.

Acrylic Resins↗
PubMed1998

Using light scattering to locate less than a microgram of protein per band in polyacrylamide tube gels after isoelectric focusing.

After separation by isoelectric focusing (IEF) or non-equilibrium pH gradient electrophoresis (NEPHGE) in 9.2 M urea polyacrylamide tube gels, proteins with Mr > 20,000 can be precipitated by shaking the gels in 25% methanol. When a fiber-optic illuminator is placed in contact with the end of the gel, the cylindrical gel transmits light by internal reflection. In regions of the gel where protein is precipitated, this light is scattered, making it visible when the gel is viewed in a darkened room against a black background. The sensitivity of the method is moderate: less than 1 microgram protein per band (in a 3-mm diameter gel) can be detected. Because the protein in the bands precipitates rapidly (30 min), the solution used (25% methanol) is fairly benign to proteins, and the apparatus is not expensive, this technique should be useful in several situations including electrophoretic purification schemes. This method is especially useful for evaluating the quality of an IEF or NEPHGE tube gel before using it for the second dimension of a two-dimensional gel.

Acrylic Resins↗
PubMed1998

Differential silver-staining sodium dodecyl sulfate-polyacrylamide gel electrophoresis: a nonisotopic method for characterizing gel-separated histone-DNA complexes.

Some nonspecific, DNA-binding proteins, like the linker histones, precipitate DNA upon binding. This is a poorly understood process that limits analysis of such nucleoprotein complexes using standard gel electrophoresis. To circumvent this problem, low concentrations of glutaraldehyde were used to crosslink the linker histones to DNA; then the partially crosslinked complexes were solubilized in SDS2 and separated by SDS-PAGE. Differential detection was accomplished using two different silver staining protocols that preferentially stained either proteins or nucleic acids. A technique was developed which allows the relative proportion of linker histones and DNAs in each detected band to be determined, and is referred to as differential staining SDS-PAGE (DS-SDS-PAGE). DS-SDS-PAGE provides a novel, non-isotopic means for characterizing multiple nucleoprotein bands separated by polyacrylamide gel electrophoresis. In applying this method to a model linker histone-DNA study, we were able to detect both protein-DNA and protein-protein contacts that are important in linker histone assembly onto DNA.

Animals↗
PubMed1998

Identification of oxidized proteins based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunochemical detection, isoelectric focusing, and microsequencing.

It has been demonstrated that an age-associated increase in protein oxidation is a highly selective rather than random phenomenon. Addition of carbonyl groups is a widely used marker for the detection of oxidative damage to the proteins. Carbonyls can be detected immunochemically using antibodies against dinitrophenylhydrazine (DNPH), a reagent that specifically reacts with protein carbonyl groups. In this paper, a procedure for the identification of oxidized proteins during aging or under pathological conditions is described. Protein samples were treated with DNPH and then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by immunochemical detection. Proteins exhibiting positive immunostain were then purified by SDS-PAGE followed by isoelectric focusing. The focused protein band was further concentrated by another run of SDS-PAGE and transferred onto Immobilon-P membrane. The identity of the protein was revealed by automated Edman microsequencing and a computer database search. This method is successfully demonstrated for the identification of housefly arginine kinase, a cytosolic protein that is involved in the energy metabolism of insect muscle cells.

Amino Acid Sequence↗
PubMed1998

Separation of radiolabeled orthophosphate and adenosine 5'-triphosphate by 20% polyacrylamide gel electrophoresis: an assay for brain microsomal Mg2+/Ca2+ ATPase activity.

Measuring orthophosphate is an important tool in biochemical analyses used to study membrane transport ATPases essential for calcium homeostasis. Current techniques involve extraction of radioactive phosphate with organic solvents, a technique that results in large quantities of hazardous radioactive waste. Other colorimetric assays are less sensitive and are complicated by interference of background absorbance from membrane tissue and unutilized ATP. This report describes a unique assay for the detection of inorganic phosphate and its application to the study of rat brain microsomal Mg2+/Ca2+ ATPase from a membrane fraction. The technique involves the separation of radioactive phosphate from unused gamma-radiolabeled ATP by resolution on 20% polyacrylamide gels. Both are visualized with X-ray film and quantitated by liquid scintillation counting after extraction from the gels. The assay can detect as little as 4.1 pmol of radiolabeled ATP and ATPase activity in 3.5 ng/microliter of membrane protein. This method offers the advantage of simultaneous quantitation of radiolabeled ATP and radioactive orthophosphate without the generation of large quantities of radioactive waste. The results demonstrate the development of a novel assay procedure for quantitating orthophosphate that is extremely sensitive, reproducible, and applicable to the study of any phosphate liberating enzyme.

Adenosine Triphosphate↗
PubMed1999

Protein purification from polyacrylamide gels by sonication extraction.

A protein purification procedure using sonication extraction from polyacrylamide gels (PAGE), which involves identification of a particular protein band and its excision, homogenization, sonication, and subsequent passage through a Sephadex G-25 minicolumn, is reported. Our results show a high degree of recovery regardless of the nature of the protein (soluble or membrane bound) or the characteristics of the gel (SDS-PAGE, native gels, or Tricine-SDS-PAGE). The percentage of recovery was dependent on the protein concentration applied in the gel. This technique is fast, gives high yield, provides good resolution, and can be used without any specialized equipment. The method was tested with a wide variety of membranes and soluble proteins and was found to give good results and to be applicable to different studies. The amino acid sequence of one of the purified proteins (Rf 0.45 stallion ejaculated sperm protein) was determined successfully.

Animals↗
PubMed1999

Mobility of acetylated histones in sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

We describe an altered mobility for acetylated histone isoforms in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Isoforms of histones H3 and H4 with a higher acetylation degree have a slightly faster electrophoretic mobility. Since acetylation neutralizes the positive charge of the epsilon-amino group of lysine, without significantly changing the molecular mass of the protein, the acetylation-dependent mobility shift could be explained by the increase of the net negative charge of the SDS-histone complexes. A possible consequence of this differential mobility for the acetylation site determination by protein microsequencing from SDS gels is discussed.

Acetylation↗
PubMed1999

A single-sample method for determination of carbohydrate and protein contents glycoprotein bands separated by sodium dodecyl sulfate- polyacrylamide gel electrophoresis.

A method is described for determination of carbohydrate and protein contents of glycoproteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then electroblotted onto polyvinylidene difluoride (PVDF) membranes. Blots were stained, and appropriate pieces of PVDF membranes were excised, destained, and subjected to sequential hydrolysis with 0.2 M trifluoroacetic acid (TFA) for 1 h at 80 degrees C, then with 2 M TFA for 4 h at 100 degrees C, and finally with 6 M HCl at 100 degrees C for 24 h to release sialic acids, neutral sugars with hexosamines, and amino acids, respectively. In some instances preliminary methanolysis was used. Carbohydrates including sialic acids were quantitated by high pH anion exchange chromatography with pulsed amperometric detection. Protein content of the bands was determined as amino acids by the fluorescamine or ninhydrin method. In the calculation of results proper adjustments were made for small amounts of fucose released by hydrolysis with 0.2 M TFA at 80 degrees C, and for partial degradation of protein during hydrolysis with 2 M TFA at 100 degrees C. Recoveries of amino acids from hydrolysates of glycoproteins that had been electroblotted onto PVDF membranes equaled those of carbohydrates. This was possible because of preliminary hydrolysis of glycoproteins with TFA, as well as washing of wet, instead of dried, PVDF membranes after hydrolysis with 6 M HCl. The two modifications increased yields of amino acids by about 30%. The method was successfully applied to the determination of molar and weight percentage composition of human transferrin, band 3 protein, glycophorin A, and alpha(1)-acid glycoprotein. In each case the results obtained for directly hydrolyzed and electrophoresed/electroblotted glycoproteins were practically identical. We also determined the glucosamine content of band 4.1 protein of erythrocytes.

Carbohydrates↗
PubMed2000

Reevaluation of the electrophoretic migration behavior of soluble globular proteins in the native and detergent-denatured states in polyacrylamide gels.

Although sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis is widely used for estimating molecular masses of proteins, considerable uncertainty still exists both about the structure of SDS-protein complexes and about their mechanism of electrophoretic migration. In this study, soluble globular proteins, with masses of 14-200 kDa, were heat-denatured in the presence of SDS and their relative total molecular volume and net charge were estimated from Ferguson plots of electrophoretic mobility vs acrylamide concentration. Native globular protein served as standards for overall molecular size and effective radii. Results revealed at least two independent electrophoretic migration mechanisms for the SDS-protein complexes: (i) for proteins in the 14-65 kDa range at <15% acrylamide, linear Ferguson plots suggested that they migrated ideally and that their effective radii could be estimated in this manner: (ii) concave plots at higher gel concentrations, and for complexes derived from larger proteins, indicated that migration in these cases could be described by reptation theory. Migration of the large proteins at lower gel concentrations and small proteins at higher gel concentrations was not well described by either theory, representing intermediate behavior not described by these mechanisms. These data support models in which all but the smallest SDS-protein complexes adopt a necklace-like structure in which spherical micelles are distributed along the unfolded polypeptide chain. Possible relations to recent alternative models of gel electrophoresis are also discussed.

Acrylamide↗