Selection of Escherichia coli and Pseudomonas putida cultures with an enhanced resistance to immobilization on polyacrylamide gel

Clones of Escherichia coli (A4, A70, G60) and Pseudomonas putida (A70, G30) with an elevated resistance to the process of immobilization in polyacrylamide gel and to the action of monomeric acrylamide were selected from the parent E. coli IBPM B115 and P. putida. The isolated cultures remained resistant to the above actions for a long time. The frequency at which cells with the elevated resistance appeared was comparable with the frequency of bacterial mutations. The plasmid analysis did not reveal the presence of plasmid DNA in the cells of the isolated cultures. The decrease in the viability index of bacterial populations caused by their immobilization in polyacrylamide gel and by the action of monomeric acrylamide did depend on the growth phase. The cells were more resistant to these actions in the stationary phase. The isolated cultures were more resistant as compared to the parent cultures irrespective of the growth phase.     

Regioselective immobilization of short oligonucleotides to acrylic copolymer gels.

Four types of polyacrylamide or polydimethyl-acrylamide gels for regioselective (by immobilization at the 3' end) of short oligonucleotides have been designed for use in manufacturing oligonucleotide microchips. Two of these supports contain amino or aldehyde groups in the gel, allowing coupling with oligonucleotides bearing aldehyde or amino groups, respectively, in the presence of a reducing agent. The aldehyde gel support showed a higher immobilization efficiency relative to the amino gel. Of all reducing agents tested, the best results were obtained with a pyridine-borane complex. The other supports are based on an acrylamide gel activated with glutaraldehyde or a hydroxyalkyl-functionalized gel treated with mesyl chloride. The use of dimethylacrylamide instead of acrylamide allows subsequent gel modifications in organic solvents. All the immobilization methods are easy and simple to perform, give high and reproducible yields, allow long durations of storage of the activated support, and provide high stability of attachment and low non-specific binding. Although these gel supports have been developed for preparing oligonucleotide microchips, they may be used for other purposes as well.     

Improved immobilization of <Emphasis Type="Italic">Lactobacillus rhamnosus</Emphasis> ATCC 7469 in polyacrylamide gel,preventing cell leakage during lactic acid fermentation

A new procedure for improved immobilization of Lactobacillus rhamnosus ATCC 7469, producing solely l(+)-lactic acid, in polyacrylamide was developed. A series of gels with varied ingredients concentrations and order of addition was prepared and were tested in batch and repeat-batch processes. Our results revealed that the crucial step for successful immobilization was the initial incubation of the cells in pure 10% AA that leads to improved entrapment in the polyacrylamide gel. In contrast, all gels derived from previously prepared stock AA/MBAA released high amount of cells and free biomass was formed. The most efficient immobilization was achieved using gel, containing L. rhamnosus, incubated in 10% AA (acrylamide) and with 1% MBAA (N,N′-methylene-bis-acrylamide) added. This gel possessed optimal permeation characteristics and at the same time, the cells were completely retained in the polymer lattice (0.03 g free biomass/l at 48 h of the batch process). In addition, it yielded highly concentrated lactic acid: the conversion ratio was about 85% without pH-control for initial lactose concentrations of up to 30 g/l. A series of additional immobilization experiments showed the potential of physicochemical interactions between the monomers of acrylamide and the cell surface of L. rhamnosus.     

Fluorography--limitations on its use for the quantitative detection of 3H- and 14C-labeled proteins in polyacrylamide gels

The suitability of fluorography for the detection of 3H- and 14C-labeled proteins on polyacrylamide gradient gels has been investigated. It was found that the absorbance of the fluorographic film image produced by a given level of radioactivity decreased as the acrylamide concentration in the gel increased. The use of Coomassie brilliant blue protein dyes to stain the gel prior to fluorography reduced the absorbance of the fluorographic image. It is concluded that quantitative fluorography can only be applied to unstained gels of a uniform acrylamide concentration.     

Penicillin amidase from E. coli. Some physicochemical properties of the enzyme incorporated into polyacrylamide gel]

The physico-chemical properties of penicillinamidase (PA) immobilized in polyacrylamide gel (IPA) were investigated. It was shown that simple incorporation of PA into polyacrylamide gel was not effective because of gradual washing out of the enzyme. The use of a complex method for the immobilization (immobilization in the presence of a linking agent) resulted in higher stability of IPA, the choice of the optimal ratio of the reagents being of paramount importance. The mechanical strength of IPA was studied in model experiments.     

A method for horizontal polyacrylamide slab gel electrophoresis

We present a simplified method of preparation of polyacrylamide gels which is totally analogous to the procedure now widely used to pour and run horizontal agarose gels. The acrylamide is poured into an open air gel mold consisting of a glass plate with a masking tape border and a comb. It is subsequently run in a submarine horizontal electrophoresis apparatus. The electrophoretic mobility and resolution of DNA fragments obtained in such gels are identical to results obtained with gels poured and run in the vertical configuration. Numerous advantages of horizontal polyacrylamide gel electrophoresis are discussed.     

Source and avoidance of the "nothing dehydrogenase" effect, a spurious band produced during polyacrylamide gel electrophoresis of dehydrogenase enzymes from yeasts

During a study of the distribution of several NAD-linked dehydrogenase enzymes in various yeasts, in which polyacrylamide gel electrophoresis, followed by activity staining with phenazine methosulfate and a tetrazolium was used, a band was frequently detected, the production of which appeared to be independent of any added substrate (the "nothing dehydrogenase" effect). It has been shown that this effect is caused by alcohol dehydrogenase acting on traces of ethanol inadvertently introduced into the system. Two sources of ethanol were identified. They were (i) the enzyme extracts, which could be freed from ethanol by gel filtration, and (ii) the acrylamide used to prepare the gel, which could be freed from ethanol by recrystallization from ethanol-free chloroform. It is suggested that the use of commercial chloroform (stabilized with ethanol) as a recrystallizing solvent is the source of ethanol contamination in commercial preparations of acrylamide.     

Determination of the molecular weight of proteins by electrophoresis in slab gels with a transverse pore gradient of crosslinked polyacrylamide in the absence of denaturing agents

The molecular weight of proteins under nondenaturing conditions can be determined through polyacrylamide electrophoresis by comparing their relative mobilities at different gel concentrations with the relative mobilities of standard proteins under the same conditions (J. L. Hedrick and A. J. Smith (1968) Arch. Biochem. Biophys. 126, 155). This work describes a procedure that eliminates the need for several gels of different acrylamide concentrations with the use of a slab gel with a transverse pore gradient of crosslinked polyacrylamide.     

Polymerizing immobilization of acrylamide-modified nucleic acids and its application

The immobilization of nucleic acids on solid supports has been widely used in the detection of DNA and other biomolecules in sensor technology. Because three dimensional (3-D) hydrogel matrixes offer significant advantages for capturing probes over more conventional two dimensional (2-D) rigid substrates and the ability to provide a solution-mimicking environment, they are becoming increasingly attractive as desired supports for bio-analysis. Acrylamide-modified nucleic acids and acrylamide monomers being polymerized directly to immobilize nucleic acids is only one-step chemical process which is not interfered by exterior surroundings, and the 3-D polyacrylamide gel fabricated by this method is not required to be activated by some labile chemical treatments. Moreover, the attachment is extremely stable to withstand the cycling process involved in the polymerase chain reaction (PCR). In this paper, the development of polymerizing immobilization of acrylamide-modified nucleic acids is reviewed, and its applications in DNA sequence high-throughput analysis including mutation analysis and the whole genome sequencing are summarized.     

Immobilization of acrylamide-modified oligonucleotides by co-polymerization.

A flexible chemistry for solid phase attachment of oligonucleotides is described. Oligonucleotides bearing 5'-terminal acrylamide modifications efficiently co-polymerize with acrylamide monomers to form thermally stable DNA-containing polyacrylamide co-polymers. Co-polymerization attachment is specific for the terminal acrylamide group. Stable probe-containing layers are easily fabricated on supports bearing exposed acrylic groups, including plastic microtiter plates and silanized glass. Attachment can be accomplished using standard polyacrylamide gel recipes and polymerization techniques. Supports having a high surface density of hybridizable oligonucleotide (approximately 200 fmol/mm2) can be produced.     

A rapid assay for assessment of sphingosine kinase inhibitors and substrates

The effect of incorporating carbon nanotubes (CNTs) in the gel matrix on the electrophoretic mobility of proteins based on their molecular weight differences was investigated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). More specifically, a reduction in standard deviation in the molecular weight calibration plots by 55% in the case of multiwalled carbon nanotubes (MWCNTs) and by 34% in the case of single-walled carbon nanotubes (SWCNTs) compared with that of pristine polyacrylamide gels was achieved after incorporating an insignificant amount of functionalized CNTs into the gel matrix. A mechanism based on a more uniform pore size distribution in CNT modified polyacrylamide gel matrix is proposed. Furthermore, the impact of SWCNTs and MWCNTs on the mobility of proteins in different molecular weight regimes at a given acrylamide concentration offers a tunable gel matrix in terms of the selection of molecular weight ranges of proteins. The robustness and excellent reproducibility of the CNT–PAGE protocol are expected to have a significant impact on the molecular weight determination of newly isolated proteins.     

Immunological identification of complex proteins resolved by sodium dodecyl sulfate polyacrylamide disc gel electrophoresis.

Immunological identification of an antigen resolved from a protein complex by sodium dodecyl sulfate polyacrylamide gel electrophoresis has been attained. The identification is based on the formation of immunoprecipitin lines after the antigen diffuses laterally from acrylamide gel transverse slices into a surrounding agarose gel. This technique was designed for study of contractile and regulatory protein complexes of non-muscle cells where the scarcity of tissue precludes easy purification or high yield of muscle-like proteins. It complements double-gel immunodiffusion or immunoelectrophoresis and its use may be extended to other protein complexes.     

Denaturing Urea Polyacrylamide Gel Electrophoresis (Urea PAGE)

Urea PAGE or denaturing urea polyacrylamide gel electrophoresis employs 6-8 M urea, which denatures secondary DNA or RNA structures and is used for their separation in a polyacrylamide gel matrix based on the molecular weight. Fragments between 2 to 500 bases, with length differences as small as a single nucleotide, can be separated using this method¹. The migration of the sample is dependent on the chosen acrylamide concentration. A higher percentage of polyacrylamide resolves lower molecular weight fragments. The combination of urea and temperatures of 45-55 °C during the gel run allows for the separation of unstructured DNA or RNA molecules.In general this method is required to analyze or purify single stranded DNA or RNA fragments, such as synthesized or labeled oligonucleotides or products from enzymatic cleavage reactions.In this video article we show how to prepare and run the denaturing urea polyacrylamide gels. Technical tips are included, in addition to the original protocol ^1,2.     

Separation of apolipoprotein B species by agarose-acrylamide gel electrophoresis

Human apolipoprotein (apo) B has been recognized to exist in two different forms designated apoB-100 and apoB-48. The two apoB forms are usually separated by NaDodSO4 gel electrophoresis with a low percentage polyacrylamide gel in a tube gel apparatus. However, the matrix of this low percentage gel is relatively weak, and one can separate the two forms of apoB in a slab gel apparatus only if one utilizes a gradient polyacrylamide gel or a higher percentage polyacrylamide gel which results in a poorer separation of the protein bands. We have developed an agarose-acrylamide gel electrophoretic method to separate the two major apoB forms. The gel is a mixture of 0.5% agarose and 2% acrylamide. The agarose-acrylamide method is fast, has the advantage of being able to be used on an analytical or preparative scale in a vertical slab gel apparatus, and the gel is of sufficient strength to be used in immunoblotting and/or radioautography.     

Bioconversion of acrylonitrile to acrylamide using polyacrylamide entrapped cells of <Emphasis Type="Italic">Rhodococcus rhodochrous</Emphasis> PA-34

The nitrile hydratase (NHase) of Rhodococcus rhodochrous PA-34 catalyzed the conversion of acrylonitrile to acrylamide. The resting cells (having NHase activity) (8 %; 1 mL corresponds to 22 mg dry cell mass, DCM) were immobilized in polyacrylamide gel containing 12.5 % acrylamide, 0.6 % bisacrylamide, 0.2 % diammonium persulfate and 0.4 % TEMED. The polyacrylamide entrapped cells (1.12 mg DCM/mL) completely converted acrylonitrile in 3 h at 10 °C, using 0.1 mol/L potassium phosphate buffer. In a partitioned fed batch reactor, 432 g/L acrylamide was accumulated after 1 d. The polyacrylamide discs were recycled up to 3×; 405, 210 and 170 g/L acrylamide was produced in 1st, 2nd and 3rd recycling reactions. In four cycles, a total of 1217 g acrylamide was produced by recycling the same mass of entrapped cells.     

Benzene degradation by bacterial cells immobilized in polyacrylamide gel

Summary Whole cells ofPseudomonas putida were immobilized in polyacrylamide gel and their ability to utilise benzene was examined. On initial immobilization cells were found to lose 40–70% of their activity. This activity could be restored by incubation in a medium containing benzene and succinate. It was also found that partial activation could be achieved by incubation with iron salts, in the absence of a carbon source. Electron microscopy showed this activation to be accompanied by an increase in cell numbers, with the formation of cell conglomerates within gel interstices. However, under some conditions, prolonged elution with substrate resulted in cell disruption and loss of activity.     

Improvements in polyacrylamide gel slicing

An improved polyacrylamide gel slicer has been devised that provides rapid uniform slicing with a precision of 4–6%. The advantages of this type of slicer are: The gel is sliced directly from the electrophoresis tube; gel diameter and length can vary with no modification of the system; and gels with a range of acrylamide concentrations can be fractionated with no pretreatment of the gel.     

Reversible covalent immobilization of ligands and proteins on polyacrylamide gels

Ligands and proteins were covalently but reversibly immobilized on polyacrylamide gels using novel acrylic monomers whose syntheses are reported here. These reagents have an acrylyl group at one end for copolymerization into gels, an N-succinimidyl ester at the other allowing rapid immobilization of molecules having an available primary amino group, and a cleavable disulfide bond in the middle. Two immobilization methods were developed using these reagents. In the first method, a ligand with a primary amino group was treated with the immobilization reagent in anhydrous ethanol and the resulting amide derivative was purified and copolymerized with acrylamide and bisacrylamide resulting in the desired reversible immobilization. In the second method, the immobilization reagents (at densities up to 50 mumol/ml) were directly copolymerized with acrylamide and bisacrylamide to form activated gels of the desired shape and porosity. Proteins or other ligands in aqueous buffers were then added to the activated gels resulting in their covalent immobilization. Ligands or proteins immobilized using the methods reported here remained stably bound even when gels were subjected to boiling in detergents or high-ionic-strength buffers. Immobilized ligands were readily released (greater than 97%) from gels by treatment with quantitative amounts of aqueous dithiothreitol (DTT) under mild conditions. Immobilized proteins were also released (up to 87%) from the gels by DTT treatment. Small ligands (e.g., aminohexyl glycosides), active enzymes, and glycoproteins were immobilized, and then recovered, using these reagents.     

Polyacrylamide gels copolymerized with active esters. A new medium for affinity systems.

A new and versatile method for linking biologically active ligands to a polyacrylamide matrix is reported. Active esters of acrylic acid (N-succinimicyl acrylate and N-phthalimidyl acrylate) were synthesized, then copolymerized with acrylamide and N,N'-methylenebisacrylamide. Displacement of the active ester in the gel thus formed by various ligands containing aliphatic amino groups resulted in the formation of stable amid bonds between the ligands and the polyacrylamide gel. The affinity gel thus prepared has the following advantages: (i) resistance to chemical and microbiological degradation, (ii) ease of control of ligand level and higher levels of ligand possible, (iii) ease of control of porosity, and (iv) total displacement of the active ester under suitable conditions. Efficacy of this system was tested by preparation of 6-aminohexyl 2-acetamido-2-deoxy-beta-D-glucopyranoside derivative polyacrylamide gel by the described method. It was found to be more effective for purification of wheat germ agglutinin than the previously published affinity chromatography systems and the wheat germ hemagglutinin was obtained in crystalline form.In addition, partial resolution of isolectins was obtained from the affinity gel witha pH gradient.     

Studies on immobilized fungal spores for microbial transformation of steroids: 11 alpha-Hydroxylation of progesterone with immobilized spores of Aspergillus ochraceus G8 on polyacrylamide gel and other matrices

Hydroxylation in the 11 alpha-position in the progesterone molecule employing immobilized spores of Aspergillus ochraceus strain No. G8 (CDRI catalogue No.) was achieved. For immobilization the activity of the spores was evaluated on a variety of matrices such as alginate beads, epoxy resin beads, polyacrylamide gel, and collagen. Spores entrapped in polyacrylamide gel were found to be the most active. Studies of various parameters, e.g. monomer content, cell loading capacity, optimum pH, temperature, and substrate concentration, were carried out on polyacrylamide gel. In polyacrylamide, the entrapped spores normal decay pattern, as indicated by loss of activity, was observed after four uses. At the end of 15 cycles, the residual activity was found to be 18% of the original. It was possible to regenerate the activity by incubating the preparation in a nutrient medium. The regenerated spores showed increasing rate of loss of activity upon recycling.