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Visualization of sialoglycoproteins in polyacrylamide gels by acidic ninhydrin reaction.

A new method for staining sialoglycoproteins in polyacrylamide gel after disc electrophoresis is described. The method utilizes the reaction of sialic acids with an acidic ninhydrin reagent which yields a stable color with an absorption maximum at 470 nm. After electrophoresis, the polyacrylamide gel is placed in a test tube and heated with 5 ml of the acidic ninhydrin reagent for 10 min in a boiling water bath. Sialoglycoproteins are detected as brown bands. No additional procedure such as destaining is necessary. When 20 micrograms fetuin, a sialoglycoprotein, per gel is applied, the band remains visible for at least 2 h. Stained gel can be scanned with a gel scanner at 470 nm. When the stained gel was dried on a sheet of polypropylene filter, the color was stable for at least one month. The present method is superior to the method using Stains-all (3,3'-diethyl-9-methyl-4,5,4',5'-dibenzothiacarbocyanine) in specificity and simplicity for the detection of sialoglycoproteins.

Electrophoresis, Polyacrylamide Gel↗

Determination of polyacrylamide in soil waters by size exclusion chromatography.

Determination of polyacrylamide (PAM) concentration in soil waters is important in improving the efficiency of PAM application and understanding the environmental fate of applied PAM. In this study, concentrations of anionic PAM with high molecular weight in soil waters containing salts and dissolved organic matter (DOM) were determined quantitatively by size exclusion chromatography (SEC) with ultraviolet (UV) absorbance detection. Polyacrylamide was separated from interferential salts and DOM on a polymeric gel column eluted with an aqueous solution of 0.05 M KH2PO4 and then detected at a short UV wavelength of 195 nm. Analysis of PAM concentrations in soil sorption supernatants, soil leachates, and water samples from irrigation furrow streams showed that SEC is an effective approach for quantifying low concentrations (0-10 mg L(-1)) of PAM in waters containing soil DOM and salts. The method has a lower detection limit of 0.02 microg and a linear response range of 0.2 to 80 mg L(-1). Precision studies gave coefficients of variation of < 1.96% (n = 4) for > 10 mg L(-1) PAM and < 12% (n = 3) for 0.2 to 3 mg L(-1) PAM.

Acrylic Resins↗

Polyacrylamide distribution in columns of organic matter-removed soils following surface application.

Knowledge of how polyacrylamide (PAM) penetrates and distributes in a soil profile after application in irrigation water is important for understanding PAM conditioning depth and evaluating its environmental effects. Little is known, however, about PAM distribution in soil because of the difficulty in quantifying PAM content in natural soils. By using a recently modified substrate-borne PAM quantification method, PAM distribution in columns of organic matter-removed soils was determined. Results showed that penetration of PAM into the soil was affected by salt level of irrigation water, soil texture, initial soil water content, water application method, and other factors. Polyacrylamide penetration depth was about one-eighth to one-half of the water penetration depth, with a particularly high PAM retention in the top few centimeters of the soil. Under different experimental conditions, the PAM retained in the top 0 to 2 cm of soil ranged from 16 to 95% of the total applied amount. More favorable solution-soil contact conditions, longer solution-soil contact time, and lower initial soil moisture caused much more PAM retention in the top few centimeters of the soil. High sorptive affinity of PAM on soil is the main reason for its low penetration into the soil. Although these results were not obtained from natural soils, they are still helpful in improving our understanding of PAM transport behavior in soils.

Acrylic Resins↗

Detection and quantitation of unlabeled nucleic acids in polyacrylamide gels.

A method for the detection and quantitation of unlabeled nucleic acids in polyacrylamide gels is presented. The technique is a variation on UV shadowing. The technique is based on the observation that some storage phosphor screens (Kodak in this case) are sensitive to UV light in the range where nucleic acids absorb most strongly. Nucleic acid present in a gel resting on the screen absorbs the UV light and casts an ultraviolet shadow on the storage phosphor screen. The amount of nucleic acid in a particular area of the polyacrylamide gel is inversely proportional to the average pixel intensity in the corresponding area of the image. The technique therefore provides an electronic record of the gel in addition to the ability to quantitate the amount of nucleic acid present in any particular band. The storage phosphor screen images are erased by visible light. If the passage of that light to the screen is blocked by the presence of solid objects or images, the reverse process can be achieved, i.e., the recording of positive images by visible light etching of a uniformly charged background. By this method, we are able to transform old autoradiographic data into digital form for easy storage, computerized manipulation and distribution.

Autoradiography↗

Evaluation of proteinuria by two-dimensional polyacrylamide gel electrophoresis after kidney transplantation.

Urinary proteins were studied in patients after kidney transplantation during various functional states (normal function, acute rejection, chronic rejection) by two-dimensional polyacrylamide gel electrophoresis. In the first dimension, the proteins were separated according to their electric charge by isoelectric focusing, and in the second dimension according to their molecular weight by sodium dodecyl sulfate polyacrylamide gel electrophoresis. After electrophoresis the proteins were visualized using a highly sensitive silver stain technique. The combination of these methods allowed the discrimination of up to 250 protein spots in human urine, most of them unidentified up to now. Functional changes of the kidney transplants were accompanied by complex changes of the protein pattern in urine. These changes cannot be simply compared to the tubular and glomerular pattern of proteinuria which can be identified by one-dimensional sodium dodecyl sulfate electrophoresis. The 2D electrophoresis of urinary proteins may develop into a useful tool in localizing of kidney damage and in the evaluation of renal disease.

Albuminuria↗

Specific staining of sialic acid components on sodium dodecyl sulfate polyacrylamide gel.

Specific staining of sialic acid components after sodium dodecyl sulfate polyacrylamide gel electrophoresis can be carried out as follows: 1) extract glycoprotein of erythrocyte membranes or serum by the phenol-saline method, 2) electrophorese the extract on 5% polyacrylamide gel containing 0.1% sodium dodecyl sulfate at constant current, 3) treat the gel with chilled 0.04 M HIO4 for 45 minutes, 4) replace the periodic acid solution with one containing resorcinol 0.6 g, conc. HCl 50 ml, 0.1 M CuSO4 0.5 ml and H2O 50 ml, 5) warm the container in boiling water until blue violet sialic acid bands become clear, 6) replace the staining solution with a mixture of equal parts water and concentrated HCl and observe at once.

Amido Black↗

A simultaneous visualization of the antioxidant enzymes glutathione peroxidase and catalase on polyacrylamide gels.

A simple and sensitive method for the simultaneous visualization of glutathione peroxidase and catalase on polyacrylamide gels is described. The procedure included: (1) running samples on a 7.5% polyacrylamide gel, (2) soaking the gel in a certain concentration of reduced glutathione (0.25-2.0 mM), (3) soaking the gel in GSH plus H2O2 or cumene hydroperoxide, (4) finally staining with a 1% ferric chloride 1% potassium ferricyanide solution. The best concentration of glutathione for simultaneous visualization of glutathione peroxidase in mouse liver homogenates and also it is specific for glutathione peroxidase since other peroxidases such as lactoperoxidase, horseradish peroxidase and glutathione S-transferase cannot be visualized. Using this method, it was found that unlike catalase, glutathione peroxidase is heat resistant (68 degrees C, 1 min), but sensitive to 10 mM sodium iodoacetate.

Animals↗

Molecular weights of three mouse milk caseins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and kappa-like characteristics of a fourth casein.

Caseins of mouse milk are phosphoproteins which precipitate at pH 4.6, stain blue with "Stains-all," and stain red with "Stains-all" following alkaline phosphatase digestion. Four caseins were separated electrophoretically in sodium dodecyl sulfate-polyacrylamide gels varying from 8.5 to 15% acrylamide. Molecular weights for three of these proteins were 43,200, 27,700, and 25,900. The molecular weights determined for bovine alphas1 and beta caseins by this method were similar to those previously obtained by other methods. A fourth mouse casein contained carbohydrate, phosphorus, and sialic acid. This protein was rennin-sensitive and behaved anomalously on sodium dodecyl sulfate polyacrylamide gels, as did bovine kappa-casein. Because of similarities with bovine kappa-casein, it was designated with "kappa-casein" of mouse milk.

Animals↗

Immunological identification of avian monomeric and polymeric immunoglobulin M and immunoglobulin A after fractionation on sodium dodecylsulfate pore gradient polyacrylamide gels.

Naturally existing variants of Immunoglobulin M (IgM) and Immunoglobulin A (IgA) in the serum of immunologically competent, as well as immunologically defective UM-B19 chickens can be detected without time consuming purification. The different molecular forms of IgM and IgA were separated by gel filtration. Pentameric IgM and dimeric IgA were well separated from the 7S-peak that contained monomeric IgM and IgA. A method for immunological identification of the protein components is described. Pore gradient gel electrophoresis was combined with antigen-antibody crossed electrophoresis in horizontal agarose slabs. The principle of the method is that IgM and IgA in their different molecular forms in the mixtures to be assayed after gel filtration are first separated with high resolution by sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis using a pore gradient of 3 to 15%. In the second step, a slab of polyacrylamide gel with the separated proteins in transferred onto agarose layers containing appropriate anti- mu- and anti-alpha antiserum and SDS in a concentration of .01%. The technique is easy to perform and gives reproducible results. In chicken serum, the monomeric state of IgM (184,000 d), along with the pentameric state (920,000 d) were identified. Serum IgA exists in dimeric (340,000 d) and in monomeric (170,000 d) states. No differences between the commercial Leghorn and dysgammaglobulinemic and normogammaglobulinemic lines were found.

Agammaglobulinemia↗

Electrophoresis of acid soluble proteins from rat liver nuclei and polysomes on large two-dimensional polyacrylamide gels.

Increased resolution of 0.4 N H(2)SO(4) soluble rat liver polysomal and nuclear proteins was achieved by initial electrophoresis on 15 percent polyacrylamide disc gels (25 times 0.5 cm) followed by electrophoresis on 16 percent polyacrylamide gel slabs (25 times 15 cm). With this method, it was found that the histones were resolved into 12 spots, the polysomal proteins A1 and A9 were each resolved into two components, and polysomal proteins A1, A1 and A1 with molecular weights of approximately 14,000 were separated from the other proteins by their rapid migration in the first dimension. Moreover, no histones were found in the acid extracts of the polysomes.

Animals↗

Detergent-mediated destaining of coomassie brilliant blue-stained SDS polyacrylamide gels.

A simple and one-step detergent-mediated destaining procedure for SDS Polyacrylamide gels for proteins is described. Suspension (5%, w/v) of a commercially available household detergent, Vim Ultra, has been found to be very efficient in destaining polyacrylamide gels without interfering with the resolution of proteins. As compared to the routinely used solvent (methanol-acetic acid-water)-mediated destaining procedure, the present method is economical and user-friendly.

Detergents↗

Isolation and purification of guinea pig inner ear antigens by preparative polyacrylamide gel electrophoresis.

OBJECTIVE: To isolate and purify the subcomponents of guinea pig inner ear antigens for further study on the autoimmunity of the inner ear. METHODS: Inner ear tissues were homogenized in phosphate buffered saline containing 0.1% SDS, and then frozen and defrosted repeatedly to extract inner ear antigens. Preparative polyacrylamide gel electrophoresis was used to separate the subcomponents of inner ear antigens. Following electrophoresis, the protein bands were localized by rapid staining and destaining. RESULTS: The major protein bands were clearly distinct when 3 mg of crude inner ear antigens was loaded, and the three major subcomponents (31, 42-45 and 60 kD proteins) accounted for about 25.99%, 21.91% and 21.10%, respectively. CONCLUSION: Preparative polyacrylamide gel electrophoresis can be used to purify the major subcomponents of inner ear antigens.

Animals↗

Fate and efficacy of polyacrylamide applied in furrow irrigation: full-advance and continuous treatments.

Polyacrylamide (PAM) is applied to 400000 irrigated hectares annually in the USA to control irrigation-induced erosion, yet the fate of dissolved PAM applied in irrigation water is not well documented. We determined the fate of PAM added to furrow streams under two treatments: Initial-10, 10 mg L(-1) PAM product applied only during the initial hours of the irrigation, and Cont-1, 1.0 mg L(-1) PAM product applied continuously during the entire irrigation. The study measured PAM concentrations in 167-m-long PAM-treated furrow streams and along a 530-m tail ditch that received this runoff. Soil was Portneuf silt loam (coarse-silty, mixed, superactive, mesic Durinodic Xeric Haplocalcid) with 1.5% slope. Samples were taken at three times during the irrigations, both during and after PAM application. Polyacrylamide was adsorbed to soil and removed from solution as the streams traversed the soil-lined channels. The removal rate increased with stream sediment concentration. Stream sediment concentrations were higher when PAM concentrations were <2 mg L(-1) a.i., for early irrigations, and when untreated tributary flows combined with the stream. In these cases, PAM concentration decreased to undetectable levels over the flow lengths used in this study. When inflows contained >6 mg L(-1) PAM a.i., stream sediment concentrations were minimal and PAM concentrations did not change down the furrow, though they decreased to undetectable levels within 0.5 h after application ceased. One percent of applied PAM was lost in tail-ditch runoff. This loss could have been eliminated by treating only the furrow advance or not treating the last two irrigations.

Acrylic Resins↗

[Analysis of ribosomal proteins and ribosomal subunits of pea seeds by electrophoresis in polyacrylamide gel].

The amino acid composition of overall protein of ribosomes and ribosomal subunits of pea seeds has been found typical of ribosomal protein. Electrophoresis in polyacrylamide gel demonstrates that proteins extracted by the solution of 3 M LiCl-4 M urea from purified ribosomes of pea seeds move towards the cathode at pH 2.2 and separate into 41 components. Electrophoresis in a tris-glycine buffer at pH 9.2 does not reveal any substance corresponding to acid proteins. Similar distribution patterns are observed when ribosomal particles are isolated with or without triton (0,5%). The treatment of ribosomes by deoxycholate results in some changes, depending on the detergent concentration. All the protein components detected in ribosomes, except one, are present in the subunits. Proteins of large and small ribosome subunits produced 26 and 21 components respectively in polyacrylamide gel electrophoresis. The distribution patterns of proteins of the two subunits appear to be different. The majority of the components of the large and small subunits differ in mobility. The data obtained suggest considerable specificity of the protein composition of 60S and 40S subunits of 80S ribosomes in higher plants.

Electrophoresis, Polyacrylamide Gel↗

[Effects of polyacrylamide on settling and separation of oil droplets in polymer flooding produced water].

The research found anion polyacrylamide (HPAM) had positive and negative effects on oil-water separation. Polymer made oily wastewater's viscosity increase and reduce rising velocity, and polymer can also increase intensity of water films between oil droplets and lengthen coalescence time of oil droplets. Those were not in favor of settling and separation for oil droplets. The positive effects on separation were that polyacrylamide had flocculating activity and made small droplets contact each other and combine into big droplets. When polymer's molecular weight was 2.72 x 10(6), and concentration was less than 800 mg/L, polymer was in favor of oil droplets settling and separation. The prime reason for oily wastewater of polymer flooding difficult to dispose was that initial median diameters of oil droplets were small. The transverse flow oil separator can intensify oil droplets combination and shorten rising time. The locale experiments showed the separator was suitable for dealing with oily wastewater of polymer flooding.

Acrylic Resins↗

[Characterization of various digestive enzyme activities of Palaemon serratus Pennant (Crustacea, Decapoda) after polyacrylamide gradient gel electrophoresis].

Soluble proteins of hepatopancreatic, extracts of Palaemon serratus (Crustacea, Deapoda) are separated by polyacrylamide gradient gel electoresis. The characterisation of the enzymatic activities is carried out directly on the polyacrylamide gel. The zymogram includes the following activities: acid and alcalin phosphatses, phosphamidase, alpha-glucosidase, beta-glucosaminidase, beta-glucuronidase and twenty bands with esterasic activities. The molecular weights are indicated.

Acid Phosphatase↗

Isoelectric focusing of human hair keratins: correlation with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns and effect of cosmetic treatments.

A new isoelectric focusing (IEF) technique in polyacrylamide gels with 6M urea and 1.5% Nonidet P40 has been developed to characterize human hair samples. The phenotypes demonstrated with this procedure has been correlated with the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns described by other authors. The method described can be applied in the forensic science analysis of a single human hair. Using the same IEF technique we have studied the changes in electrophoretic patterns of cosmetically treated hair. The characteristics of the modifications observed and its utility in forensic science work are also discussed in this paper.

Detergents↗