Two methods that facilitate autoradiography of small 32P-labeled DNA fragments following electrophoresis in agarose gels

Two methods which permit detection by autoradiography of small 32P-labeled DNA fragments resolved by agarose gel electrophoresis are described. Agarose gel electrophoresis poses problems for autoradiography as (i) the gels are normally too thick to allow autoradiography without being dried first, and (ii) fragments of DNA of 1000 bp or less in length are readily lost during drying. In this study DNA fragments as small as 121 bp have been retained in agarose gels upon drying. This has been achieved by either (i) first fixing the DNA with the cationic detergent cetyltrimethylammonium bromide, or (ii) drying the agarose gels onto Zeta-Probe charge-modified membranes.     

A simple and reliable method for the drying of polyacrylamide slab gels

A simple method for the drying of polyacrylamide slab gels is described. 2-mm thick gels with gradients of 5–20% acrylamide dry without complications. The dried gels are transparent permitting transmission densitometry and fluorography.     

Immobilization of a lipoxygenase in silica gels for application in aqueous media

The encapsulation of soybean lipoxygenase-1 (LOX-1) in silica gels and its application in an aqueous medium, were studied. The main silica precursor was tetramethoxysilane (TMOS) but the introduction of hydrophobic SiCH₃ groups brought with methyltrimethoxysilane (MTMS) was evaluated. Other sol–gel synthesis parameters investigated comprised partial or complete drying by evaporation and CO₂ supercritical drying. The influence on LOX-1 activity of the various chemicals with which the enzyme was in contact during encapsulation (acetone, methanol, polyvinyl alcohol), as well as the temperature and pH, were examined. The activity of free and encapsulated LOX-1 was assayed on the oxygenation reaction of linoleic acid by dioxygen from air dissolved in aqueous medium, in a UV–vis spectrophotometer. With free LOX-1, the reaction advancement could be followed in continuous in the spectrophotometer. With the gels, in a first approach, the conversion was simply determined after 15 min reaction after filtration of the liquid, to discriminate between active and inactive gels. For the most interesting gels, the kinetics were then assessed by continuous recording in the UV spectrophotometer, after placing a small piece of gel (≈15 mg) directly in the cell. The best gels had an activity ≈30% of free LOX. The present studies, supplemented by characterization of the gels texture and structure, respectively by nitrogen adsorption and ²⁹Si MAS NMR, showed that drying a gel before use in aqueous media was detrimental to the activity. This effect is due to a contraction of the gel network which occurs when a dry aerogel sample is dipped in water after drying. Hence gels containing LOX-1 enzyme must not be dried but kept in water impregnated state, for optimum use.     

Structure and properties of silk fibroin-poly(vinyl alcohol) gel

A series of porous silk fibroin-poly(vinyl alcohol) blend gels were prepared from corresponding aqueous mixtures by freeze- or air-drying, and their structure and mechanical properties were examined. The air-dried gels had higher crystallinity and much greater strengths than the freeze-dried gels, and therefore are suitable for mechanical uses. The freeze-dried gels had characteristic porous structure potentially useful as cell culture substrate. Its structure could be systematically varied by changing freezing temperature and freeze-thaw pretreatments before drying.     

Kinetic models for the dynamical behavior of polyacrylamide (PAAm)–κ-carrageenan (κC) composite gels

A fluorescence method was employed for studying the drying and swelling of PAAm–κC composite gels, which were formed from acrylamide (AAm) and N, N’- methylenebisacrylamide (BIS) with various κ–carrageenan (κC) contents by free radical crosslinking copolymerization in water. Composite gels were prepared at 80 °C with pyranine (Py) as a fluorescence probe. Scattered light, I_sc, and fluorescence emission intensities, I_em, were monitored during drying and swelling of these gels. The fluorescence intensity of pyranine increased and decreased as drying and swelling time are increased, respectively, for all gel samples. The Stern–Volmer equation combined with moving boundary and Li-Tanaka models were used to explain the behavior of I_em during drying and swelling processes respectively. It is found that the desorption coefficient D_d decreased as κC contents were increased for a given temperature during drying. However, the cooperative diffusion coefficient, D_s presented exactly the opposite case. Conventional gravimetrical and volumetric experiments were also carried out during drying and swelling of PAAm–κC composite gels. It was observed that D_d and D_s values measured with the fluorescence method were found to be much larger than they were measured with the conventional methods.     

电泳凝胶板干燥前的预处理

本文强调了电泳后凝胶板干燥前预处理的重要作用,并介绍了几种预处理的方法。     

电泳凝胶板干燥前的预处理

本文强调了电泳后凝胶板干燥前预处理的重要作用并介绍了几种预处理的方法。     

Preparation of chitin/cellulose composite gels and films with ionic liquids

In this study, we performed preparation and characterizations of the chitin/cellulose composite gels and films using the two ionic liquids, 1-allyl-3-methylimidazolium bromide and 1-butyl-3-methylimidazolium chloride. First, chitin and cellulose were dissolved in each appropriate ionic liquid. Then, the two liquids were mixed in the desired ratios at 100 °C to give the homogeneous mixtures. The gels were obtained by standing the mixtures for 4 days. On the other hand, the films were obtained by casting the mixtures on glass plates, followed by soaking in water and drying. The obtained gels and films were characterized by XRD and TGA measurements. The mechanical properties of the gels and films were evaluated under compressive and tensile modes, respectively.     

Rapid polyacrylamide slab drying using a microwave gel dryer

A new gel dryer which uses microwave energy instead of radiant heat to dry slab electrophoresis gels has been designed. The use of microwaves results in substantial decreases in drying time. The potential utility of this instrument is discussed.     

An improved staining technique for precipitin bands in agar or agarose gels

A method for the staining of proteins in agar and agarose gels using three stains simultaneously and a mordant is described. When compared with conventional Coomassie brilliant blue R-250 staining procedures, it requires a comparable time expenditure but has the following advantages: 1) it is threefold to fourfold more sensitive; 2) there is increased photographic resolution on conventional photographic material; and 3) the stain has a long shelf-life and does not fade under normal lighting conditions. Conditions for the washing and drying of gels are discussed.     

A hybridization method for autoradiographic visualization of small unique sequence fragments resolved on polyacrylamide gels

A useful method is described for sequence-specific visualization of small nucleic acid fragments on polyacrylamide gels. Excess highly radioactive probe is annealed in solution to smaller fragments. The resulting hybrids are trimmed (under appropriate conditions) with S1 nuclease to yield duplex fragments whose sizes are determined by the strand contributed by the smaller unlabeled fragment, but which also contain a labeled strand from the radioactive probe. These fragments can then be resolved on polyacrylamide gels and visualized by drying the gel and subjecting it to autoradiography. This method is shown to visualize fragments as small as 40 base pairs.     

Chitin and carbon aerogels from chitin alcogels

Supercritical point drying of gels is a common technique for the production of a specific category of nano-porous materials called aerogels. We have successfully prepared chitin aerogels by extracting the solvent from the alcogels (gels with an alcohol as the solvent) with carbon dioxide under supercritical conditions. The produced nano-porous materials exhibit the typical properties of aerogels such as high porosity, high surface area, and low density, which make them quite attractive for many applications. The use of chitin, however, is of particular interest for the production of aerogels not only for being abundant and cheap but also because it has important inherent properties such as biocompatibility, non toxicity, thermal and chemical stability. In this work we examine the influence of different parameters on the porosity characteristics of the aerogels, such as the drying conditions (temperature and pressure), the nature of the solvent, and the gel concentration. Since these aerogels collapse in liquid medium, we also investigated the possibility of their utilization as carbon aerogel precursors.     

Measurements of elastic moduli of silicone gel substrates with a microfluidic device

Thin layers of gels with mechanical properties mimicking animal tissues are widely used to study the rigidity sensing of adherent animal cells and to measure forces applied by cells to their substrate with traction force microscopy. The gels are usually based on polyacrylamide and their elastic modulus is measured with an atomic force microscope (AFM). Here we present a simple microfluidic device that generates high shear stresses in a laminar flow above a gel-coated substrate and apply the device to gels with elastic moduli in a range from 0.4 to 300 kPa that are all prepared by mixing two components of a transparent commercial silicone Sylgard 184. The elastic modulus is measured by tracking beads on the gel surface under a wide-field fluorescence microscope without any other specialized equipment. The measurements have small and simple to estimate errors and their results are confirmed by conventional tensile tests. A master curve is obtained relating the mixing ratios of the two components of Sylgard 184 with the resulting elastic moduli of the gels. The rigidity of the silicone gels is less susceptible to effects from drying, swelling, and aging than polyacrylamide gels and can be easily coated with fluorescent tracer particles and with molecules promoting cellular adhesion. This work can lead to broader use of silicone gels in the cell biology laboratory and to improved repeatability and accuracy of cell traction force microscopy and rigidity sensing experiments.     

Influence of the porous texture of silica gels on the enzymatic activity of lipases in esterification reactions

Pseudomonas Cepacia lipases were encapsulated in hybrid silica-polyvinyl alcohol gels, which were dried either supercritically in order to form aerogels or by evaporation so as to obtain xerogels. In each case, the catalytic activity of the encapsulated enzymes was studied and compared to free enzyme biocatalysis. This study demonstrates that the activity of the enzyme is increased when the procedure used allows it to resist capillary stresses occurring during the drying of the gel. That is, esterification rates are higher when the gels are synthesized with a base catalyst, such as NaF, in the presence of polyvinyl alcohol and then dried supercritically.     

中枢神经蛋白质组分析中双向电泳技术的建立

建立和优化了中枢神经组织蛋白质组分析所需的双向电泳及相关技术.由于中枢神经组织结构的特殊性,样品处理非常困难.对样品液组成、样品处理、上样方式、上样量、IPG胶条和SDS-聚丙烯酰胺凝胶电泳染色方法和保存等相关技术进行了比较研究和条件优化后,以固相pH梯度等电聚焦为第一向和SDS均一胶（T=12.5%）的水平电泳为第二向,成功地得到了神经组织双向电泳图谱.     

From hydrocolloids to high specific surface area porous supports for catalysis

Polysaccharide hydrogels are effective supports for heterogeneous catalysts. Their use in solvents different from water has been hampered by their instability upon drying. While the freeze-drying process or air-drying of hydrocolloid gels led to compact solids with a low surface area, drying the gel in CO2 beyond the critical point provided mesoporous materials with a high specific surface area. Their effectiveness as a support for catalysis was exemplified in the reaction of substitution of an allyl carbonate with morpholine catalyzed by the hydrosoluble Pd(TPPTS)3 complex. The influence of water on the catalytic activity and the properties of the support was evidenced.     

Drying and storage of polyacrylamide slab gels: a simple procedure

A simple procedure for drying and storing of polyacrylamide slab gels is described. A polyacrylamide slab gel is fixed in acetic acid plus glycerol and then sandwiched between a gel bond plastic sheet and a dialysis membrane in the presence of a minute amount of gelatin and dried on the benchtop at room temperature. The fixed gel can be stored indefinitely.     

Polyacrylamide gel miniaturization improves protein visualization and autoradiographic detection

Polyacrylamide gels shrink to one-quarter of their original area when soaked in a 50% (w/v) solution of polyethylene glycol. Gel miniaturization improves the contrast of protein bands, with four valuable consequences. (i) A 5- to 10-fold increase in sensitivity for Coomassie blue is observed. (ii) Gels are more durable; i.e., they resist tearing when wet and they do not crack during drying under vacuum. (iii) Shrunken gels give sharper photographic images and provide better interlane protein band comparisons. (iv) Condensed protein bands lead to an increased sensitivity for detecting low-abundance, radioactively-labeled proteins by fluorography.     

Polyacrylamide gel electrophoresis on micro slabs

An electrophoretic technique using micro polyacrylamide flat gels is described and its usefulness demonstrated. The gels are vertically cast and electrophoresed in slab form (75 × 18 × 0.75 mm) in closed thin glass cells (cuvets) made from detachable microscope slides. The main features of the method are: requirement of small sample quantities (0.1–20 μg contained in <1–5 μl solution), simultaneous analysis of several samples in a single gel, relatively brief running periods, easy removal of the gel for rapid staining due to the two-piece gel mold, little pattern diffusion, convenient optical evaluation, drying, autoradiography and other contact print methods, and easy application of immunodiffusion techniques. Continuous gradient gels can be prepared. The advantage and complications of the technique are discussed and certain applications in biochemistry, clinical chemistry, and medicine are pointed out.     

FTIR spectroscopy of NH3 on acidic and ionotropic alginate aerogels

The acidity of alginate aerogel films has been investigated by infrared spectroscopy of adsorbed NH(3). Supercritical drying of the alginate provided samples with a surface area of several hundred square meters per gram, in which the probe molecule could reach all acidic sites. Free carboxylic groups were studied on acid-gelled alginates and were found to behave as effective Br?nsted acid sites. Ionotropic alginate gels formed by alkaline earth cations presented only the Lewis acidity of the cations. Ionotropic gels formed by transition metal cations presented both Lewis and Br?nsted sites, because of the presence of a fraction of free carboxylic groups. The incomplete salification was correlated to the pH of the gelling solutions.