A simple and rapid method for the isolation of peptides from sodium dodecyl sulfate-containing polyacrylamide gels

A rapid and simple method is described for the recovery of peptides from sodium dodecyl sulfate-containing polyacrylamide gels. It involves the electrophoretic concentration of a peptide in the stacking gel followed by elution into glycerol. The method requires no special equipment or chemicals, and the elution can be made using the same electrophoretic systems used in the separation step. The method is more rapid than normal extraction procedures, and simpler than most electrophoretic elution methods described. The method can be used for isolation of microgram as well as milligram quantities of an individual peptide with yields of approximately 100%.     

Solid-phase extraction and high-performance liquid chromatography analysis of lipoxygenase pathway products

Liquid chromatography methods for quantitation of leukotrienes and HETEs (hydroxyeicosatetraenoic acids) in biological samples are described. Extraction is accomplished by acetonitrile precipitation of proteins followed by selective acetonitrile elution from a solid-phase C18 extraction cartridge. Isocratic elution of extracts from short reverse-phase columns with 3 microns C18-bonded silica results in elution of all components of interest in less than 10 min. The addition of the mobile-phase additives, trifluoroacetic acid and triethylamine, enable the peptido-leukotrienes to be eluted with excellent peak shape and unique elution times. As little as 1 pmol of each metabolite can be detected by uv spectrophotometry with a wavelength change from 280 to 235 nm midway through the chromatographic run. This method is demonstrated by the extraction and analysis of leukotriene and HETE standards from human serum.     

Novel purification of carbonic anhydrase III from human, rat and baboon muscle

A new method of inhibitor elution from DEAE cellulose is described for carbonic anhydrase III. Highly purified fractions free of other isozymes were obtained after one column elution.     

Simplified techniques for elution of proteins from polyacrylamide gel, staining, destaining, and isoelectric focusing

A simple and rapid electrophoretic method for the simultaneous elution of proteins in high yields from polyacrylamide gel slabs is described. The apparatus is simple and easily constructed. The method involves vertical elution from a horizontally placed gel across its thickness into buffer-soaked polyurethane foam. Even the slow-moving, high-molecular-weight protein ferritin is eluted in 1 h. The technique can also be used for rapid destaining as well as for simultaneous staining and destaining of the polyacrylamide gel which are completed in 15 and 30 min, respectively. Polyurethane foam strips have also been used to simplify the preparative isoelectric focusing technique and the subsequent elution of protein bands, obviating the need for Ultrodex, fractionating grid, sample applicator, and elution columns.     

Automated derivatization with o-phthalaldehyde for estimation of amino acids in plasma using reversed-phase high performance liquid chromatography

A fully automated method for quantitative estimation of plasma amino acids using fluorescence detection of ophthaladehyde/2-mercaptoethanol derivatives of the analytes and their separation by gradient elution reversed-phase HPLC has been described. The method is simple and the three-step gradient elution is suitable for routine analysis of a large number of biological samples due to clear resolution, high degree of precision, accuracy, cost-effectiveness and lack of interference from chemical contaminations. Using this method, 19 amino acids were completely resolved and the within-run coefficients of variation ranged from 2.53 to 10.7% with a mean variation of 5.68%.     

Chromatography on DEAE-cellulose microcolumns: A quantitative method for the fractionation of small quantities of glycosaminoglycans

A simple, sensitive, and efficient method is described for qualitative and quantitative determination of glycosaminoglycans (GAG) synthesized by embryonic mouse teeth. After release from proteoglycan aggregates by enzymatic treatment, a mixture of different GAG was absorbed on a DEAE-cellulose microcolumn (Whatman DE-52 microgranular) at low salt concentration. The different types of GAG were eluted by stepwise increases in the concentration of NaCl. Glycopeptides, which generally contaminate the extract, can be completely removed prior to the elution of GAG. The eluate fractions were analyzed by rechromatography on the same column, using gradient elution. The stepwise elution is suitable for analysis as well as preparation of labeled GAG, the supply of which is limited in amount. The scale of chromatography can easily be stepped up. Quantitative analysis of GAG from embryonic mouse teeth is presented to demonstrate the usefulness of this method.     

An improved treatment of data for estimation of molecular weights by sephadex gel filtration

A method for analysis of elution data of proteins, obtained from Sephadex gel filtration experiments, is described. The relevant elution data from seven different proteins, with known molecular weights and Stoke's radii, were fitted into various equations relating elution parameters and molecular size parameters. It was observed that polynomial relationships represented elution data for proteins with a much greater degree of precision than linear equations. The validity of this procedure was also checked by analysing gel filtration data available in the literature and it was concluded that a better fit was obtained using polynomial relationships, provided a sufficiently large number of experimental points were available for numerical analysis. Using this method, values of 320,000 ± 7000 for the molecular weight, and (60 ± 0.4) × 10^?8 cm for the Stoke's radius of Neurospora NAD-specific glutamate dehydrogenase were calculated.     

A simple and sensitive method for the quantitative estimation of collagen.

A method to quantitate collagen in solution is described. It is based on binding of the dye Sirius Supra red F3BA by collagen followed by elution and estimation of the bound dye in a spectrophotometer. The assay can be used in the range from 10 to 100 μg protein. The method is rapid, specific, simple, sensitive, and highly reproducible.     

The resolution of five linker histone subtypes from chicken erythrocytes

A method is described for the isolation of 4 highly purified H1 linker histone subfractions, and histone H5, from chicken erythrocytes. Fractionation is achieved under non-denaturing conditions by elution from CMC-25 Sephadex with an NaCl gradient. When applied to calf thymus H1, the order of subtype elution differs from that acheived on Amberlite IRC-50 [(1966) J. Biol. Chem. 241, 5790-5797; (1976) Biochemistry 15, 4233-4242]. The two column types employed in series could provide a means for improving subtype purity.     

Isolation of maltopentaose from corn syrup by chromatography on granulated hydroxylapatite.

A method is described for the isolation of maltopentaose from corn syrup based upon elution from granulated hydroxylapatite with increasing concentrations of water in ethanol.     

Plate height determination for gradient elution chromatography of proteins

A method for determining the plate height HETP from the elution curve obtained by the linear gradient elution (LGE) ion-exchange chromatography (IEC) of proteins is presented. The method was developed on the basis of the numerical solutions of a chromatography model which considers the zone sharpening and the distribution coefficient as a function of the salt concentration. The plate height HETP is determined from the peak width and the salt concentration at which the peak is eluted in LGE. The method was applied to the experimental results with various ion-exchange chromatography media. A calculation example based onthe present method is presented to show how the chromatographic and operating parameters should be tuned to obtain a desired resolution. A simplified calculation procedure for the peak profile is also described. (c) 1995 John Wiley & Sons, Inc.     

Ion exchange chromatography of proteins-predictions of elution curves and operating conditions. II. Experimental verification

The applicability and validity of the model developed in Part I were confirmed experimentally. In this article, various proteins were eluted both by stepwise and linear gradient elution on DEAE ion exchangers under a variety of experimental conditions. Adsorption isotherms were measured as a function of ionic strength in batch experiments. The moment method was empolyed for the determination of various parameteres such as the gel-phase diffusion coefficient and the longitudinal dispersion coefficient. By use of these parameters and the experimentally measured ionic strength of the peak position, the number opf plates was determined according to the method described in Part I. Theoretical elution curves were calculated with the experimentally measured adsorption eqluilibria and the number of plates. Good agreement was observed between theory an experiments. Various factors affecting the separation were investigated. It was found that the effect of the number of plates for salts, N'(p), was negligible except the case of stepwise elution of high ionic strength buffer. When elution curves were symmetrical, the widths of the elution curves were inversely proportional to the square root of the number of plates of proteins, N(p), as in other chromatographic techniques. A simple graphical method for prediction of the peak position in linear gradient elution described in Part I was found applicable when the elution curves were symmetrical. A useful correlation of prediction of the peak width in a linear gradient elution was proposed on the basis of the approximate solution derived in Part I of this study. This graphical method and correlation permit easy prediction of the peak position and peak width in linear gradient elution in the case of symmetrical elution curves.     

Purification of ribonuclease T1

An improved method for purifying ribonuclease T1 from Aspergillus oryzae is described. The method uses gradient elution from DEAE-cellulose and sulfopropyl-Sephadex columns followed by gel filtration on Sephadex G-50 to give almost 100 mg (50% yield) of ribonuclease T1 from 100 g of starting material in less than 5 days.     

Quantitation of proteins bound to polyvinylidene difluoride membranes by elution of coomassie brilliant blue R-250

A rapid and simple method for the quantitation of stained proteins bound to polyvinylidene difluoride (PVDF) membranes via the elution of Coomassie brilliant blue R-250 is described. A mixture of standard proteins was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotted onto PVDF membranes. Spectrophotometric analysis of dye eluted from protein bands in the range of 0.5-10 micrograms gave a linear change in the absorbance at 595 nm. Maximal absorbance readings were attained following 5 min of dye elution, and the readings remained unchanged for elution times up to 60 min. The method requires no unusual reagents or equipment, is suitable for the analysis of multiple samples, and does not consume the protein in the process of quantitation. This technique provides a useful means for the quantitation of proteins bound to PVDF membranes prior to amino acid sequence determination, immunological analysis, or other biochemical characterizations.     

Studies in cryoprotection: I. A simple method for the controlled introduction and removal of cryoprotective agents during organ perfusion

A method is presented for the introduction and removal of cyroprotective agents from kidneys, utilizing the principle of gradual addition and elution during continuous perfusion. The method differs from those described previously in that it utilizes a standard perfusion machine (Water MOX 100) and a motorized syringe pump; both of which are widely available in laboratories involved in clinical and experimental organ preservation. In addition, a simple method for measuring vascular resistance is described which relies on a comparison of flow rates through the perfused organs to that through a fixed resistance. The utility of this approach is illustrated by studies of organ function and vascular resistance.     

Concentration of proteins using carboxymethyldextran

Carboxymethyldextran was used to displace several proteins from DEAE-cellulose in highly concentrated form, and the concentrated protein fractions were examined directly by gel electrophoresis. The concentration of DNA-dependent RNA polymerase II demonstrates the application of the method to a labile enzyme. The concentration of goat γ-globulin by sharp elution from CM-cellulose with carboxymethyldextran is also described.     

One-step fractionation of neutral and acidic glycosphingolipids by high-performance liquid chromatography

A procedure for a simultaneous separation of ganglioside components and neutral glycolipid components by high-performance liquid chromatography was described. One column packed with DEAE-derivatized controlled-pore glass (DEAE-CPG) was serially connected to two columns of underivatized, controlled-pore glass (CPG). A mixture of gangliosides and neutral glycolipids were loaded on DEAE-CPG and eluted with a mixture of chloroform-methanol-water, with increasing methanol and water (the first-phase gradient elution), followed by elution with increasing concentrations lithium acetate from 0.015 to 0.1 M in a mixture of chloroform-methanol-water (the second-phase gradient elution). Neutral glycolipids, mono- to hexaglycosylceramides , were separated within 80 min of the first-phase gradient elution, and mono- to tetrasialosylgangliosides were separated during the second-phase gradient elution within 60 min. The method has been applied to the determination of glycolipids isolated from rat tissues, and the procedure was found to be highly reproducible.     

Purification of commercial preparations of NADP+ from AMP contamination

A method for purification of commercial preparations of NADP+ from AMP contamination is described. The purification procedure includes one-step anion-exchange chromatography on Dowex-1 (formate) and results in a highly purified salt-free coenzyme with a yield of 70-80%. The chromatography conditions have been selected allowing for complete separation of AMP from NADP+ in a HCOOH concentration gradient. This is followed by NADP+ elution with 1.5 M HCOOH containing HCOOK at a concentration at which the salt remains in solution during subsequent precipitation and washing of NADP+ with acetone. An addition of HCOOK is necessary to reduce the coenzyme elution volume that is important for further precipitation of NADP+ with acetone.     

A rapid method for separation and assay of radiolabeled mucopolysaccharides from cell culture medium

A method is described for the separation and assay of radiolabeled glycosaminoglycuronans (GAGs) from cell culture medium. Following papain digestion and precipitation with cetylpyridinium chloride onto cellulose ester filter membrane, hyaluronic acid can be separated from sulfated GAGs by selective elution with HCl.     

A hydroxylapatite batch assay for quantitation of cellular DNA damage.

A batch elution procedure is described for quantitative measurement of DNA damage. The technique is based on alkaline unwinding of cellular DNA and separation of singlestranded from duplex forms by step elution from hydroxylapatite with phosphate formamide. The method is rapid, permits large numbers of samples to be handled simultaneously, and consistently yields recoveries of >95% of total chromatographed DNA. Because as many as 1 × 10⁷ cells per batch may be analyzed, quantitation of the eluted DNA by nonradioactive methods is feasible. The method is standardized with respect to the unwinding constant β, the alkaline DNA unwinding unit Mng, and the DNA-damaging efficiency of ionizing irradiation.