Exponential gradient maker using a disposable syringe

With a simple modification, any disposable syringe can become a reliable and easy to use exponential gradient maker. The modification consists of two notches, made with a razor blade, in the borders of the rubber sealing tip of the plunger. A clamp in the tube connected to the syringe allows control over solution flow. With the clamp prohibiting drainage, the body of the syringe is filled with the desired volume of starting solution I. A magnetic stir bar, small enough to spin inside the syringe is included. The notched plunger is introduced until no air space remains. This forms the fixed volume, closed mixing chamber, while the rest of the volume of the syringe forms the open chamber. The two chambers are connected through the notches in the plunger. The ending solution II is poured after the introduction of the plunger. Opening the clamp allows solution I in the closed chamber to flow out, and the solution II in the open chamber flows through the notches and mixes with solution I. This exponential gradient maker can be reused many times, but the low cost of the components makes it potentially disposable. This feature is especially useful when using toxic chemicals, or when pouring polyacrylamide gradient gels, since the apparatus may be disposed of after contamination or eventual polymerization.     

Pouring and Running a Protein Gel by reusing Commercial Cassettes

The evaluation of proteins using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis is a common technique used by biochemistry and molecular biology researchers^1-4. For laboratories that perform daily analyses of proteins, the cost of commercially available polyacrylamide gels (˜$10/gel) can be considerable over time. To mitigate this cost, some researchers prepare their own polyacrylamide gels. Traditional methods of pouring these gels typically utilize specialized equipment and glass gel plates that can be expensive and preclude pouring many gels and storing them for future use. Furthermore, handling of glass plates during cleaning or gel pouring can result in accidental breakage creating a safety hazard, which may preclude their use in undergraduate laboratory classes. Our protocol demonstrates how to pour multiple protein gels simultaneously by recycling Invitrogen Nupage Novex minigel cassettes, and inexpensive materials purchased at a home improvement store. This economical and streamlined method includes a way to store the gels at 4°C for a few weeks. By re-using the plastic gel cassettes from commercially available gels, labs that run frequent protein gels can save significant costs and help the environment. In addition, plastic gel cassettes are extremely resistant to breakage, which makes them ideal for undergraduate laboratory classrooms.     

Problems encountered in measuring erythrocyte pyrimidine 5'-nucleotidase activity

An improved method of two-dimensional electrophoresis that allows unequilibrated first-dimension gels to be loaded and electrophoresed on second-dimension gels twice the length used in the O'Farrell technique has been developed. Normally, the electrophoresis of unequilibrated first-dimension gels on long second-dimension gels with the resolving gel set at pH 8.8 results in poor resolution of low-molecular-weight proteins. Adjusting the pH of the resolving gel to pH 8.3 maintains the low-molecular-weight proteins in a stacked configuration during their migration through the length of the 10% acrylamide gel. Utilization of a 10 to 20% exponential polyacrylamide gradient in a resolving gel set at pH 8.3 separates these low-molecular-weight proteins with excellent resolution. Electrophoretic resolution of protein spots in resolving gels set at pH 8.8 is not as sharp as in gels set at pH 8.3, and resolution progressively deteriorates in gels set at higher pH values.     

Preparations of continuous gradient gel slabs: a simple technique.

A simple technique is described which allows casting of continuous-pore gradient gel slabs without any special equipment. The gels are not linear but satisfactory for all practical purposes. These gels compare favorably with gels made with gradient mixers and with gels obtained commercially, as has been shown by electrophoresis of standard proteins.     

Nuclear steady-state RNA from chicken immature red blood cells. Distribution of globin-coding and poly (A) sequences

Nuclear steady-state RNA and polysomal RNA of chicken immature red blood cells were isolated and separated on formamide sucrose gradients. For comparison the distribution of 9 S globin mRNA was investigated by gradient centrifugation of 125I-labelled mRNA. The material was either pooled into two fractions (less than 20 S; greater than 20 S) and translated in an Ehrlich ascites cell-free system or each gradient fraction was analyzed by hybridization with [3H]-poly (U) or [3H]-labelled DNA complementary to purified 9 S globin mRNA (globin cDNA). In neither case could evidence be obtained for the existence of a high molecular weight RNA as a probable globin mRNA precursor. Further analysis was performed by electrophoresis of RNA on exponential polyacrylamide gels in formamide and subsequent hybridization with cDNA. The results are consistent with those of gradient centrifugation and demonstrate that the distribution of globin-coding sequences in nuclear steady state RNA corresponds to that of cytoplasmic 9 S globin mRNA.     

An improved silver staining procedure for schizodeme analysis in polyacrylamide gradient gels.

A simple protocol is described for the silver staining of polyacrylamide gradient gels used for the separation of restriction fragments of kinetoplast DNA [schizodeme analysis of trypanosomatids (Morel et al., 1980)]. The method overcomes the problems of non-uniform staining and strong background color which are frequently encountered when conventional protocols for silver staining of linear gels are applied to gradient gels. The method described has proven to be of general applicability for DNA, RNA and protein separations in gradient gels.     

Polyacrylamide gels with modified cross-linkages

The retention of low molecular weight proteins during electrophoresis through gradient polyacrylamide gels was improved when a gradient of N,N′,N″-triallyl citric triamide (TACT) was superimposed on the gradient of acrylamide and N,N′-methylenebisacrylamide (MBA). Gels cross-linked only with N,N′-(1,2-dihydroxyethylene)bisacrylamide (DHEBA) are soluble in dilute periodic acid or dilute aqueous solutions of bases. DHEBA cross-linked gradient gels have a smaller pore structure at high acrylamide concentrations and a more open structure at low acrylamide concentrations than gels cross-linked with MBA. Proteins labeled with tritium and carbon-14 were fractionated through DHEBA cross-linked gradient gels and the isotopes measured after solution of the gel with periodic acid. The mild solubilizing conditions enhanced isotope resolution. The characteristics of several cross-linking molecules are discussed and reasons advanced for the superiority of those with acrylamido end groups.     

不同浓度和梯度的SDS-PAGE胶对双向电泳中蛋白分离的影响

目的：探讨双向电泳中不同浓度和梯度的SDS-PAGE胶对肠道菌蛋白分离效果的影响。方法：制备弗氏2a志贺菌2457T野生株37℃晚期全菌蛋白质样品,进行不同浓度及梯度的SDS-PAGE,研究分离肠道菌蛋白最适宜的SDS-PAGE胶浓度。结果：获得了3个不同浓度（10%、12.5%和15%）的均一胶电泳图谱和3个不同梯度（4%～15%、10%～20%和12%～14%）的电泳图谱,并比较了这些图谱的分离效果;同时,为了分析肠道菌天然表达蛋白的相对分子质量范围,鉴定了8个极端相对分子质量蛋白。结论：对于肠道菌蛋白质的分离来说,12.5%的均一胶或12%～14%的梯度胶较为适宜。     

Mouse brain protein composition during postnatal development: an electrophoretic analysis

Changes in the concentrations of mouse brain proteins during postnatal maturation were characterized by a combination of subcellular fractionation and electrophoresis. Sodium dodecyl sulfate gel electrophoresis revealed changing protein concentrations in fractions enriched in nuclei, mitochondria plus synaptic endings, microsomes and cytosol. Postnatal maturational changes in protein concentrations were most pronounced in fractions of purified myelin membranes. The use of exponential gradient gels resulted in increased resolution of low molecular weight myelin proteins. Nuclei treated with Triton X-100 exhibited no change in relative histone concentrations during brain maturation. Nonnuclear contamination of untreated nuclear fractions was shown to be a potential source of erroneous interpretations. These findings are discussed in terms of genetic products and sodium dodecyl sulfate polyacrylamide gel electrophoresis resolution.     

Agarose slab-gel electrophoresis equipment.

Simple slab-gel molds which utilize the electrophoresis apparatus described by F. W. Studier (J. Mol. Biol.79, 237 (1973)) have been designed for pouring and running agarose slab-gels. Analytical gels in which many samples are run simultaneously facilitate the assay of many enzymes which lead to physical changes in DNA, whereas the preparative gels allow the separation of large quantities (1–20 mg) of DNA fragments.     

体外构建组织工程化软骨的初步研究

观察用藻酸钠凝胶在体外长期培养软骨细胞的情况,为求进一步用藻酸钠凝胶在体外构建组织工程化软骨提供初步研究.将传代培养的、细胞终浓度为1×107/mL的软骨细胞复合藻酸钠凝胶,注入圆柱形模具中,将模具分别浸入100、200、300 mmol/L的固化剂氯化钙溶液中,固化15 min使其成形,于体外培养2、4、6周后,行HE染色、阿尔新蓝染色,了解软骨细胞的生长情况.2、4、6周时软骨细胞与藻酸钙凝胶复合良好并能在其中保持活性及分裂能力,且在6周时出现类软骨变化.因此藻酸钠凝胶是可用于体外构建组织工程化软骨的生物材料.     

Production of polyacrylamide gradient gels for the electrophoretic resolution of lipoproteins.

We describe a protocol to cast nondenaturing polyacrylamide gradient gels (SFBR3/31) for the size resolution of lipoproteins. The protocol yields gels with minimal lot-to-lot variation in length and electrophoretic properties. Absorbance profiles of cholesterol-stained lipoproteins in baboon sera were used to estimate the relative amounts of stain in four lipoprotein size classes (VLDL+LDL, HDL1, HDL2, and HDL3). When compared with gels from a commercial source, the SFBR3/31 gels gave very similar results in terms of precision (coefficients of variation) and of estimated amounts of lipoproteins in the four size classes. In other studies, we estimated peak diameters of protein-stained human lipoproteins after calibrating the gels with size standards. Peak diameters estimated using SFBR3/31 gels were highly correlated (r2 = 0.99, n = 33) with those estimated using gels from a commercial source. We conclude that the protocol reliably produces gradient gels that are suitable for the analysis of lipoprotein phenotypes.     

Isoelectric focusing in immobilized pH gradients: Principle,methodology and some applications

A new technique for generating pH gradients in isoelectric focusing is described, based on the principle that the buffering groups are covalently linked to the matrix used as anticonvective medium. For the generation of this type of pH gradient in polyacrylamide gels, a set of buffering monomers, called Immobiline (in analogy with Ampholine), is used. The pH gradient gels are cast in the same way as pore gradient gels, but instead of varying the acrylamide content, the light and heavy solutions are adjusted to different pH values with the aid of the Immobiline buffers. Available Immobiline species make it possible to generate any narrow linear pH gradient between pH 3 and 10. The behaviour of these types of gradients in isoelectric focusing is described.Immobilized pH gradients show a number of advantages compared with carrier ampholyte generated pH gradients. The most important are: (1) the cathodic drift is completely abolished; (2) they give higher resolution and higher loading capacitu; (3) they have uniform conductivity and buffering capacity; (4) they represent a milieu of known and controlled ionic strenght.     

A method for easily customizable gradient gel electrophoresis

Gradient polyacrylamide gel electrophoresis is a powerful tool for the resolution of polypeptides by relative mobility. Here, we present a simplified method for generating polyacrylamide gradient gels for routine analysis without the need for specialized mixing equipment. The method allows for easily customizable gradients which can be optimized for specific polypeptide resolution requirements. Moreover, the method eliminates the possibility of buffer cross contamination in mixing equipment, and the time and resources saved with this method in place of traditional gradient mixing, or the purchase of pre-cast gels, are noteworthy given the frequency with which many labs use gradient gel SDS-PAGE.     

Polyacrylamide slab gel electrophoresis: an improved design

An apparatus for polyacrylamide slab gel electrophoresis is decribed which combines all parts into one integral unit. It eliminates several steps in the process of sealing, pouring, and setting the gels. Construction is easy with modest workshop facilities and the design easily adapted to suit most requirements. The apparatus provides a high degree of versatility and is suitable for use with many slab gel electrophoretic techniques.     

Gel gradient electrophoresis, isoelectric focusing and two-dimensional techniques in horizontal, ultrathin polyacrylamide layers

An ultrathin layer, horizontal polyacrylamide gel system for electrophoresis, isoelectric focusing and two-dimensional techniques is described. Gel slabs 240 micron thin for unidimensional, or 360 micron thin for two-dimensional runs are cast on cellophane foils as support. The sample is loaded in pockets pre-cast in the gel (2--3 microliter size) or in trenches for two-dimensional experiments. The second dimension is routinely performed in concave exponential gel gradients, spanning an acrylamide concentration from 4% to 22.5%. The sensitivity with the common Coomassie Blue stain is very high, well below 0.1 microgram protein/band. Zymogram detections can be developed within a few minutes, thus retaining the band sharpness of the focused zones or of the bands separated in pore gradient electrophoresis. Sample handling, staining and destaining and gel drying and storage are greatly simplified and performed in a fraction of the time needed for conventional, thick gels in the 1-2 mm thickness range.     

Rectified Cell Migration on Saw-Like Micro-Elastically Patterned Hydrogels with Asymmetric Gradient Ratchet Teeth

To control cell motility is one of the essential technologies for biomedical engineering. To establish a methodology of the surface design of elastic substrate to control the long-range cell movements, here we report a sophisticated cell culture hydrogel with a micro-elastically patterned surface that allows long-range durotaxis. This hydrogel has a saw-like pattern with asymmetric gradient ratchet teeth, and rectifies random cell movements. Durotaxis only occurs at boundaries in which the gradient strength of elasticity is above a threshold level. Consequently, in gels with unit teeth patterns, durotaxis should only occur at the sides of the teeth in which the gradient strength of elasticity is above this threshold level. Therefore, such gels are expected to support the long-range biased movement of cells via a mechanism similar to the Feynman-Smoluchowski ratchet, i.e., rectified cell migration. The present study verifies this working hypothesis by using photolithographic microelasticity patterning of photocurable gelatin gels. Gels in which each teeth unit was 100–120 µm wide with a ratio of ascending:descending elasticity gradient of 1:2 and a peak elasticity of ca. 100 kPa supported the efficient rectified migration of 3T3 fibroblast cells. In addition, long-range cell migration was most efficient when soft lanes were introduced perpendicular to the saw-like patterns. This study demonstrates that asymmetric elasticity gradient patterning of cell culture gels is a versatile means of manipulating cell motility.     

Electrophoretic separation of large proteoglycans in large-pore polyacrylamide gradient gels (1.32-10.0% T) and a one-step procedure for simultaneous staining of proteins and proteoglycans.

A procedure is described for the preparation of 1.32-10% polyacrylamide gradient gels. Loose polyacrylamide gel on the top side of the gradient was stabilized with a layer of 0.4% agarose gel which also formed sample wells. The upper limit of separation achieved in these gels was estimated to be approximately 2 X 10(6) using globular protein standards. However, large aggregating proteoglycans from cartilage which have a molecular weight range of 1-4 X 10(6) penetrate and separate in these gels. A simple one-step procedure is also described for simultaneous staining of proteins and large proteoglycans in polyacrylamide gels.     

Protein staining and pH gradient determination on the same gel in isoelectric focusing.

The apparent isoelectric point of a component focused on polyacrylamide gels is normally estimated by extrapolating a pH gradient determined on one gel to another gel which has been stained for protein in order to locate the position of the component (1). The pH gradient is determined by slicing the gel transversely and reading the pH of the eluate after soaking the segments for 1–2 hr in a small amount of degassed water. It is assumed that the gradients in both gels are identical. Alternatively, an antimony microelectrode has been used to measure pH gradients directly in unsectioned gels (2). Similar techniques have been applied to polyacrylamide gel slabs and are reviewed by Vesterberg (3). Righetti and Drysdale (4) have recently reviewed these and other aspects of isoelectric focusing in gels.I report here a very precise method for the determination of a protein “isoelectric point” that can be accomplished with a single gel. The technique is demonstrated with yeast phosphoglycerate kinase and the very low density lipoprotein (VLDL) fraction from human plasma.     

Fluorographic detection of [3H]thymidine-labeled deoxyribonucleic acid using agarose gels infused with 2,5-diphenyloxazole prior to electrophoresis

A fluorographic procedure for the detection of [3H]thymidine-labeled deoxyribonucleic acids electrophoresed in agarose gels was developed. 2,5-Diphenyloxazole (PPO) was added to the agarose solution before pouring of the gel for electrophoresis. This procedure did not interfere with the electrophoretic mobility of the DNA molecules. The radioactive detection efficiency was found to be improved over an existing procedure whereby the agarose gel was infused with PPO after electrophoresis with the aid of acetic acid.