Gel Molecular Sieve Research Articles

How It All Began: A Personal History of Gel Electrophoresis.

Arne Tiselius' moving boundary electrophoresis method was still in general use in 1951 when this personal history begins, although zonal electrophoresis with a variety of supporting media (e.g., filter paper or starch grains) was beginning to replace it. This chapter is an account of 10years of experiments carried out by the author during which molecular sieving gel electrophoresis was developed and common genetic variants of two proteins, haptoglobin and transferrin, were discovered in normal individuals. Most of the figures are images of pages from the author's laboratory notebooks, which are still available, so that some of the excitement of the time and the humorous moments are perhaps apparent. Alkaline gels, acidic gels with and without denaturants, vertical gels, two-dimensional gels and gels with differences in starch concentration are presented. The subtle details that can be discerned in these various gels played an indispensable role in determining the nature of the change in the haptoglobin gene (Hp) that leads to the polymeric series characteristic of Hp 2/Hp 2 homozygotes. Where possible the names of scientific friends who made this saga of gel electrophoresis so memorable and enjoyable are gratefully included.

Microfluidic Western blotting of low-molecular-mass proteins.

We describe a microfluidic Western blot assay (μWestern) using a Tris tricine discontinuous buffer system suitable for analyses of a wide molecular mass range (6.5–116 kDa). The Tris tricine μWestern is completed in an enclosed, straight glass microfluidic channel housing a photopatterned polyacrylamide gel that incorporates a photoactive benzophenone methacrylamide monomer. Upon brief ultraviolet (UV) light exposure, the hydrogel toggles from molecular sieving for size-based separation to a covalent immobilization scaffold for in situ antibody probing. Electrophoresis controls all assay stages, affording purely electronic operation with no pumps or valves needed for fluid control. Electrophoretic introduction of antibody into and along the molecular sieving gel requires that the probe must traverse through (i) a discontinuous gel interface central to the transient isotachophoresis used to achieve high-performance separations and (ii) the full axial length of the separation gel. In-channel antibody probing of small molecular mass species is especially challenging, since the gel must effectively sieve small proteins while permitting effective probing with large-molecular-mass antibodies. To create a well-controlled gel interface, we introduce a fabrication method that relies on a hydrostatic pressure mismatch between the buffer and polymer precursor solution to eliminate the interfacial pore-size control issues that arise when a polymerizing polymer abuts a nonpolymerizing polymer solution. Combined with a new swept antibody probe plug delivery scheme, the Tris tricine μWestern blot enables 40% higher separation resolution as compared to a Tris glycine system, destacking of proteins down to 6.5 kDa, and a 100-fold better signal-to-noise ratio (SNR) for small pore gels, expanding the range of applicable biological targets.

Effectiveness of Desiccant Coated Regenerative Wheels from Transient Response Characteristics and Flow Channel Properties—Part II: Predicting and Comparing the Latent Effectiveness of Dehumidifier and Energy Wheels Using Transient Data and Properties

In this paper, a new transient response theory is developed to predict the moisture or water transfer time constant and the fully developed flow moisture effectiveness of dehumidifier wheels and latent energy effectiveness of energy wheels. A new method to experimentally determine the latent energy or water transfer time constant is presented. This analytical model, which uses the new empirical time constant and its theoretical model, is then corrected for entrance length, carryover, sorption temperature changes, and manufacturing variations. The model permits the designer of these wheels and the HVAC applications engineer the opportunity to explore how each flow channel property and wheel operating condition changes the effectiveness. The theoretical latent and sensible effectiveness of energy wheels with molecular sieve and silica gel coatings are determined for two energy wheels, and these results are compared with the effectiveness calculated using experimental data at steady state ARI testing conditions. The comparisons are in agreement within the uncertainty bounds. The analytical model is also used to predict the latent effectiveness for two similar energy wheels where it is assumed that the desiccant coatings are interchanged. This comparison shows that the new hypothetical silica gel coating on the energy wheel would be more effective than the new molecular sieve coating on an energy wheel for the transfer of water vapor. The moisture transfer effectiveness is predicted for two dehumidifier wheels with the same design and desiccants as the tested energy wheels (i.e., the molecular sieve wheel and silica gel wheel). These wheels, which are used for supply air drying, are operated with the same ARI supply air condition and 5 rpm. Considering the comparisons for energy wheels, the molecular sieve and silica gel coatings are predicted to result in similar performance factors but with much lower effectiveness. This reduction in moisture effectiveness for the dehumidifier wheels is caused mostly by the reduction in wheel speed. Further research is required to investigate the performance of dehumidifier wheels at lower wheel speeds.

Lead inhibits the core protein synthesis of a large heparan sulfate proteoglycan perlecan by proliferating vascular endothelial cells in culture

We characterized proteoglycans synthesized by growing cultured bovine aortic endothelial cells after exposure to lead. Lead significantly decreased the incorporation of both [ 3H]glucosamine and [ 35S]sulfate into glycosaminoglycans accumulated in the cell layer and the conditioned medium of the cells in a dose-dependent manner. Proteoglycans metabolically labeled with [ 35S]sulfate in the presence of lead were separated into heparan sulfate proteoglycans (HSPGs) and more highly charged chondroitin/dermatan sulfate proteoglycans by DEAE-Sephacel ion-exchange chromatography. It was found that lead markedly inhibited the synthesis of HSPGs. Sepharose CL-4B molecular sieve gel filtration showed that the marked decrease by lead occurred in the high molecular weight subclass of HSPGs. However, the length of heparan sulfate chains (approximately 50 kDa) was unchanged when analyzed by Sepharose CL-6B chromatography. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis of core proteins showed that lead reduced the accumulation of a high molecular weight (approximately 400 kDa) HSPG core protein in the cell layer and the conditioned medium; the core protein was identified as a perlecan core by Western blot analysis. It is suggested that lead inhibits the synthesis of the perlecan core protein in growing endothelial cells without a change of heparan sulfate chain length. The present data support the hypothesis that inhibition of endothelial cell proliferation by lead may result from a lower response of the cells to endogenous basic fibroblast growth factor whose binding to the receptor is strongly promoted by heparan sulfate chains of perlecan.

Thrombin Reduces Large Heparan Sulfate Proteoglycan Molecules in Cultured Vascular Endothelial Cell Layers through Inhibition of Core Protein Synthesis

We investigated the alteration of heparan sulfate proteoglycans induced by thrombin in cultured vascular endothelial cells. Heparan sulfate proteoglycans, which were metabolically labeled with [ 3H] glucosamine and [ 35S] sulfate, were isolated by DEAE-Sephacel ion-exchange chromatography and characterized by molecular sieve gel filtration. Core proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [ 35S] amino acids-labeled heparan sulfate proteoglycans after digestion with hepartinase. It was revealed that the high molecular weight subclass of heparan sulfate proteoglycans in the cell layer was markedly decreased by thrombin without changes of the hydrodynamic size of the molecules and the molecular weight of heparan sulfate chains. In addition, thrombin decreased the amount of large heparan sulfate proteoglycan core protein with a molecular weight of approximately 400 kDa, probably perlecan core, in the cell layer and the conditioned medium. The present data suggest that thrombin-induced decrease in the amount of heparan sulfate in vascular endothelial cell layer includes a reduction of the number of large heparan sulfate proteoglycan perlecan molecules through a suppression of the core protein synthesis.

Amino acids and peptides. XXX. Preparation of Arg-Gly-Asp (RGD) hybrids with poly(ethylene glycol) analogs and their antimetastatic effect.

Hybrids of a fibronectin-related peptide[Arg-Gly-Asp (RGD)] with poly(ethylene glycol) (PEG) analogs were prepared by a simple and easy procedure. Two amino-PEG analogs were used as carriers for hybrid formation of the RGD. One was poly(oxyethylene)dipropylamine and the other was Jeffamine ED type, which has branched chains. RGD peptides were formed stepwise on PEG analogs by the diisopropylcarbodiimide method. The synthetic intermediates were easily purified by molecular-sieve gel chromatography and the final products were purified by molecular-sieve gel chromatography, followed by HPLC. This simple and easy preparation procedure using molecular-sieve gel chromatography for purification of synthetic intermediates is advantageous for the preparation of peptide-polymer hybrids. We found that PEG is stable to HF treatment at 0 degree C for 1 h. The inhibitory effect of the RGD hybrids on experimental metastasis of B16-BL6 was examined in mice. The Jeffamine type hybrid showed no inhibitory effect at the dose of 1 mg/mouse, but poly(oxyethylene)dipropylamine type hybrid was inhibitory at the same dose. The effect of the latter hybrid was about the same as that of 1 mg of RGD. One mg of the hybrid contains 0.18 mumol of RGD and 1 mg of RGD is 2.38 mumol. Thus it can be said that the inhibitory effect of RGD was potentiated by hybrid formation with poly(oxyethylene)diisopropylamine.

Binding of sodium dodecyl suplhate to an integral membrane protein and to a water-soluble enzyme : Determination by molecular-sieve chromatography with flow scintillation detection

We have determined the binding of sodium dodecyl (SDS) to the human red cell glucose transporter (polypeptide, M r 54 117) and to a water-soluble enzyme, N-5′-phosphoribosylanthranilate isomerase—indole-3-glycerol-phosphate synthase (PRAI—IGPS) from Escherichia coli ( M r 49 484). [ 35S]SDS was equilibrated with each protein on molecular-sieve chromatography at a series of SDS concentrations. The binding ratios of SDS to protein were determined by flow scintillationdetection and automated amino acid analyses. Unexpectedly the glucose transp an automated amino acid analyses. Unexpectedly the glucose transporter, which is a transmembrane protein, bound about the same amount of SDS per gram of protein as did the enzyme. At 1.6 m M SDS, slightly below the critical micelle concentration (CMC) (1.8 m M) in the eluent, the binding ratio was 1.6 g SDS/G protein for both the glucose transporter and PRAI—IGPS. At 2.0 m M SDS (above the CMC) the glucose transporter showed a binding ratio of 1.7 g SDS/g protein. The corresponding value for the enzyme was about 1.5 g/g. The SDS—glucose transporter complex seems to be more compact than the SDS—enzyme complex as judged by molecular-sieve chromatography and by SDS—polyacrylamide gel electrophoresis. Tecent neutron scattering results have shown a protein-decorated triple-micelle struct scattering results have shown a protein-decorated triple-micelle structure for the SDS—PRAI—IGPS complex. Hypothetically, the more compact SDS—glucose transporter complex may therefore consist of a dual-micelle structure. The molecular-sieve gel beads bound considerable amounts of SDS. The SDS binding to gel matrix and to the proteins increased with increasing SDS concentration up to at least 1.6–2.0 m M SDS. In the case of the water-soluble enzyme a shoulder was observed in the binding curve at 1 m M SDS, probably reflecting a change in the conformation of the complex.

Partial purification and some properties of spore germination promoter(s) for Dictyostelium discoideum excreted by Klebsiella aerogenes.

Spore germination promoter(s) for the cellular slime mold Dictyostelium discoideum was partially purified from the culture medium of Klebsiella aerogenes using Amberlite XAD-2 and molecular sieve gel column chromatography. The active components were found both outside and inside of a dialyzing bag and were separated into several fractions on a Sephadex G-75 gel column. The treatment of partially purified promoter(s) with NaIO4 oxidation resulted in the complete loss of the activity. The promoter(s) was very unstable against heat treatment at pH 12. Several enzymes, such as proteinase K, RNase, DNase, lipase, and zymolyase could not inactivate the promoter(s). Interestingly, the spore germination depended on phosphate ion at 25–100 mm and proteose peptone or yeast extract in addition to the promoter(s).

Transforming growth factor type alpha in normal human adult saliva

For the first time, transforming growth factor alpha (TGF-α) has been detected in the saliva of healthy adults. In saliva, the concentrations of TGF-α and epidermal growth factor (EGF) were determined by radioimmunoassay (RIA) to be 1–6 μg/1 saliva and 0.5–2 μg/1 saliva, respectively. The salivary TGF-α was partially purified about 1200-fold by an ethanol extraction, Bio-Gel P-30 molecular sieve chromatography and reverse-phase high-performance liquid chromatography (HPLC). At least three forms of TGF-α, with molecular weights of 6 kDa or less, were detected with molecular sieve gel filtration chromatography. A major chromatography peak, which constituted 20–30% of the extract, was a 5.6 kDa human TGF-α (hTGF-α) molecule by using RIA, radioreceptor assay (RRA), and mitogenic activity assay techniques. Reverse-phase HPLC purification further confirmed the identity of TGF-α. Our findings suggest, in contrast to what was earlier assumed, that TGF-α is present in normal adult saliva and we hypothesize that it may play a significant role in mediating proliferation and other functions of epithelial cells in the alimentary system.

Isolation and partial characterization of an ion channel protein from human sperm membranes

Human sperm cells were fractionated and plasma membrane proteins were separated by molecular gel sieving chromatography (Sephacryl S-200 followed by HPLC). A poreforming protein was extracted from sperm cell membranes. The partially purified protein migrated with M r 100,000–110,000, as determined by molecular sieving gel chromatography, and with a M r 90,000 when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. The channel activity was also extracted with Triton X-114, suggesting a hydrophobic nature for this protein. This protein was incorporated into planar lipid bilayers, resulting in the formation of voltage-dependent ion channels. Single channel fluctuations of 130 pS/unit in 0.1 m NaCl were resolved; however, channels preferentially aggregated in triplets having an open state life-time that persisted for several seconds. The channels studied here were more selective for monovalent cations than anions, but also showed some permeability to anions and larger electrolytes, suggesting a large functional pore diameter. The role of this sperm channel in normal sperm physiology and/or fertilization is presently unclear.

Seasonal variation of the hemagglutinating activities in the red alga Gracilaria verrucosa.

Three different hemagglutinins, of which the molecular weight was about 300-400K, 100K and 45K respectively, were found in the extract from a red alga Gracilaria verracosa by means of molecular sieve gel chromatography. 300-400K and 100K hemagglutinins showed the isoelectric point of pI 4.2, and agglutinated horse erythrocytes but not those of goose. On the other hand, 45K hemagglutinin had the iso-electric point of pI 4.7 and agglutinated both horse and goose erythrocytes. Hemagglutinating activities in the algal extract were found during the period of November to next June, and not in July and August. Hemagglutinins with higher molecular weight (300-400K and 100K) and lower isoelectric point (pI 4.2) were detectable in December, February and March but little or nothing in November, while 45K hemagglutinin was detected in all test periods (No-vember to next March).

Purification and some properties of intracellular esterase from Pseudomonas fluorescens.

A novel esterase was found in Pseudomonas fluorescens cells and purified to homogeneity as determined by polyacrylamide gel electrophoresis. The esterase was extracted from the cells by freeze-thawing and hypotonic treatment. Purification was achieved by ammonium sulfate precipitation, followed by successive chromatographies on DEAE-cellulose and benzylamine-agarose and then electrophoresis. The enzyme catalyzed the hydrolysis of methyl esters, such as methyl butyrate, but its hydrolyzing activity decreased with increase in the chain length of the alcohol moiety, and it did not catalyze the hydrolysis of triacylglycerols, such as triacetin. In contrast, the enzyme acted on various acyl residues in a series of methyl esters, such as dimethyl succinate, methyl methacrylate, and dimethyl malate. The optimum pH for activity of this enzyme with methyl butyrate was 7.0-8.5. The enzyme was inhibited by phenylmethylsulfonylfluoride. Its molecular weight was estimated as 48,000 by molecular sieve electrophoresis and gel filtration on Sephadex G-150.

Effect of pancreatic extracts on the faecal excretion and on the serum concentration of cobalamin and cobalamin analogues in cystic fibrosis

A malabsorption of crystalline labelled cobalamin is observed in 100% of cystic fibrosis patients. Using radioisotope dilution assays and molecular sieve gel chromatography, we determined the serum concentration and the faecal excretion of cobalamin and cobalamin analogues in nine cystic fibrosis children before and after 4 days' interruption of pancreatic extract treatment. On chromatography, the unsaturated cobalamin binders of the faecal extracts eluted in two positions with molecular masses of 44 300 and 20 300, corresponding mostly to partially degraded R binders. The amounts of the less degraded form of R binder (molecular mass 44300) increased significantly after interruption of the treatment. The cobalamin concentration in the serum remained normal after interruption of the treatment but the analogue concentrations in the serum decreased and faecal excretion of cobalamin and analogues increased significantly. These results allowed us to suggest that (1) pancreatic insufficiency in cystic fibrosis is responsible for a decrease in the absorption of digestive analogues induced by a defective degradation of R binders, and (2) cobalamin analogues have a short half-life in blood.

Determination of dissociation constants and ligand specificity of detergent solubilized surface membrane immunoglobulin a from MOPC-315

Surface membrane immunoglobulin from MOPC-315 plasmacytoma cells (smM315) was isolated by nonionic detergent lysis of radioiodinated cells and affinity chromatography on Dnp-aminohexyl-Sepharose 4B. Verification of the solubilized molecule as an integral membrane protein, distinct from secreted MOPC-315 IgA (M315) was accomplished by NaDodSO − 4-PAGE, charge-shift electrophoresis and molecular sieve gel filtration with NP-40 and deoxycholate. smM315 was compared to reduced and alkylated monomeric secreted immunoglobulins from MOPC-315, MOPC-460, and XRPC-25 by quantitative affinity chromatography (QAC) using two differently substituted Dnp-aminohexyl-Sepharose 4B resins. Unique patterns of cross-reactivity of all secreted myeloma proteins were independently established with a competitive hapten inhibition assay using 125I-Dnp 26BSA as the precipitating probe. After derivation with dinitrobenzylsulfonate, Dnp-aminohexyl-Sepharose 4B was modified with succinic anhydride which, with the inclusion of 0.03% Doc in a PBS and 0.1% NP-40 buffer, prevented nonhapten specific protein-matrix interactions during QAC. Dissociation constants determined by QAC for three ligands, (dinitrophenyl-glycine, trinitrophenyl-amino-caproate and tetramethylrhodamine) were essentially the same for smM315 and M315. Both of the other nitrophenyl binding IgA myelomas had distinct and significant differences in dissociation constants. Thus, for a differentiated antibody secreting cell which has undergone a heavy chain class switch, such as MOPC-315, the cell surface immunoglobulin has an identical ligand binding active-site as the secreted immunoglobulin.

Isolation of the antigen-active components from leptospiral serovar-specific lipopolysaccharide antigen by mild acid hydrolysis.

The serovar-specific main antigen (TM antigen) from Leptospira interrogans serovar kremastos strain Kyoto was mildly hydrolyzed With 2 N formic acid or 0.5 N sulfuric acid. Both treatments yielded an immunologically actvie component which was able to pass through a Diaflo membrane filter. The compound obtained by formic acid hydrolysis was shown to be more homogeneous by molecular sieve gel chromatography than that obtained by sulfuric acid hydrolysis. The molecular weight of the former compound was estimated to be approximately 3, 000 daltons. The compound consisted mainly of carbohydrates, and it lost almost all of its fatty acids and proteins. The substance lost its immunoprecipitable activity but showed serovar-specific inhibitory potency in a radio-immunoassay system.

Large Scale Preparation of Pure Hog Kidney Renin

The complete purification of renin raises difficult problems due to its extremely low concentration in kidney (less than 1/50,000 of total proteins). The complete purification of hog kidney renin has been realized on a large scale, starting from 300 kg of fresh hog kidneys. 14.6 mg of pure renin were obtained with an overall yield of 4%. The purification procedure involved 14 steps. The enzyme was extracted at pH 3.5. Subsequent purification steps were performed in the presence of protease inhibitors to decrease renin proteolysis. These steps included an ammonium sulfate precipitation and a batch-chromatography on DEAE-cellulose. The major purification step was an affinity chromatography on Sepharose-hexamethylene-diaminopepstatin. The enzyme obtained was further purified by molecular sieving gel filtration and isoelectric focusing.

Serum-derived immunosuppressive substances. I. Partial purification and range of action.

Two alpha-globulin fractions which had in vivo and in vitro immunosuppressive activity were prepared from human serum. The larger fraction eluted from a molecular sieving gel in the albumin fraction and was heat labile whereas the smaller one was present in the elute fraction containing materials of 8,000-25,000 daltons and was heat stable. It is believed that the smaller of the two fractions is the immunoregulatory alpha-globulin described by several other investigators. The comparative activities of various starting materials and of partially purified derivatives in several systems (in vivo and in vitro) were examined. The possibility that these substances are lymphocytic chalones is considered.

Partial purification and characterization of RTG-2 fish cell interferon

Interferon produced by rainbow trout gonadal cells (RTG-2) was partially purified. The physical, chemical, and biological properties of this in vitro produced fish cell interferon were studied. Purification was achieved by ultracentrifugation, molecular sieve gel chromatography, ion exchange chromatography, and polyacrylamide gel electrophoresis. The isoelectric point of RTG-2 interferon, as determined by CM-Sephadex (C-50) chromatography, was 7.1. Filtration through Sephadex G-150 showed that RTG-2 interferon had a molecular weight of 94,000. The partially purified material was not sedimented at 105,000 times g for 2 h at 4 C. The fish cell interferon was non-dialyzable and exhibited heat and pH stability. The partially purified material was inactivated by treatment with trypsin or 2-mercaptoethanol, but was resistant to treatment with deoxyribonuclease or ribonuclease. RTG-2 interferon which was induced by infectious pancreatic necrosis virus exhibited antiviral activity against challenge with infectious hematopoietic necrosis virus or infectious pancreatic necrosis virus. Partially purified RTG-2 interferon exhibited greater species specificity than the crude material.

Authentication of the cholinergic system in the cattle tick, Boophilus microplus

Acid-aqueous and organic-solvent extracts of organophosphate-susceptible larval cattle ticks yielded a biologically active substance which co-chromatographed with [ 14C]acetylcholine (ACh) on a molecular-sieve gel, and migrated identically with [ 14C]ACh during paper electrophoresis. The response of the rat fundus and the frog rectus abdominis muscle to these extracts was abolished after they were incubated with the specific ACh-hydrolysing enzyme, acetylcholinesterase (acetylcholine hydrolase, E.C. 3.1.1.7), or subjected to alkaline hydrolysis. Extracts of the isolated brains of adult ticks also showed biological activity which co-electrophoresed identically with [ 14C]ACh. No comparable amounts of an active substance, other than that which moved coincident with [ 14C]ACh, was found in the extracts. In terms of ACh chloride equivalents the ACh content of larvae was estimated by bioassay as 12.6 μg. per g. and that of adult brains as 47 μg. per g. Consistent with the model for the operation of the cholinergic system, and with the hypothesis for the lethal action of organophosphates, the ACh content of larvae treated with a lethal dose of Coumaphos increased significantly.

Gas Chromatography as Applied to Nuclear Technology – II. Analysis of the Hydrolysis Products of Uranium Carbides and Thorium Carbides

Gas chromatography was used for qualitative and quantitative determination of the products of the hydrolysis of uranium carbides and of thorium carbides. The products were separated on four columns: 5A molecular sieve, di2-ethylhexyl sebacate, tricresyl phosphate, and silica gel modified with squalane. The hydrocarbons were identified by use of known gaseous and liquid alkanes, alkenes, and alkynes. The numerical data were obtained by IBM 7090 computation. (auth)