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.     

小麦高分子量麦谷蛋白亚基分离方法的研究

小麦高分子量麦谷蛋白亚基（ＨＭＷ－ＧＳ）与小麦面包烘烤质量和面粉的加工特性密切相关,ＳＤＳ－ＰＡＧＥ是其常用的分离方法之一。ＳＤＳ－ＰＡＧＥ方法一般分为２类：第一类采用１１％和５％浓度的胶,后者用于分离２亚基和２＾＊亚基,该种方法常使用碱性提取液,需要２次电泳过程,且在５％浓度的胶中ＨＭＷ－ＧＳ易于和麦醇蛋白混淆;另外一类ＳＤＳ－ＰＡＧＥ采用梯度胶,配合使用银染方法,制梯度胶则使用梯度仪及磁力搅拌     

Fluorography of tritium-labelled proteins in immunoelectrophoresis

A method is described for detecting 3H-labelled proteins in immunoelectrophoretic systems performed on agarose gels. The method is based on the incorporation of a polyacrylamide gel into the agarose gel after the electrophoresis. This mixed gel has the characteristics of a polyacrylamide gel, making it possible to use fluorography as has been described for polyacrylamide gels. The applicability of the fluorography method is demonstrated by analyzing 3H-labelled human serum albumin and 3H-labelled pig intestinal brush border proteins by quantitative immunoelectrophoresis.     

Alignment of two-dimensional electrophoresis gels

Two-dimensional electrophoresis is a major separating technique for proteins in proteomics. Alignment of gel images is critical for intra-laboratory or even more difficult inter-laboratory gel comparisons. In the paper, we propose a novel iterative closest point (ICP) method for 2D-gel electrophoresis image alignment. The paper seeks to introduce an information theoretic measure as one part of distance metric to gel image alignment. We combine intensity information of spots with geometric information of landmarks by applying information potential idea. The proposed method has been applied to both synthetic and real gel images accessible in public 2D-electrophoresis gel protein databases. The high accuracy and robustness of the algorithm indicate that it is promising for gel image alignment.     

A new approach for on-line enrichment in electrophoresis of dilute protein solutions

A method is described for on-line enrichment/zone sharpening of a sample of negatively charged proteins (an analogous method for cationic proteins can be designed). The sample is applied on the top of a 5-mm thick layer of a neutral polyacrylamide gel which rests on another 5-mm thick, large-pore polyacrylamide gel which contains positively charged groups. The latter gel layer is attached to the neutral gel column, used for the electrophoretic separation of the proteins. When a voltage is applied the proteins start migrating and become electrostatically adsorbed at the top of the charged, large-pore gel layer (pH 5.4). With the upper electrode vessel filled with a buffer of a pH higher (pH 7.7) than that employed in the enrichment step and with a voltage between the electrodes, these enriched proteins are released (because the enrichment gel is non-charged at pH 7.7) with zone sharpening and migrate into the 5-cm long column (i.d. 5 mm) of a neutral, large-pore polyacrylamide gel for electrophoretic analysis. Upon the electrophoretic migration from the enrichment gel into the separation gel a second zone sharpening may occur, if the increase in pH from 5.4 to 7.7 in the separation gel is not close to momentary. By employing colored test proteins the efficiency of the enrichment step is visually illustrated by a picture. The principle of the concentration method described has been employed also in chromatographic experiments and can with appropriate modifications also be used in other electrophoretic methods, such as capillary electrophoresis.     

A method for quantifying radioactivity associated with protein in silver-stained polyacrylamide gels.

A method is described in which individual proteins labeled with weak beta-emitting radionuclides, separated by polyacrylamide gel electrophoresis, and stained with silver are released from the gel by the use of the periodate soluble cross-linking agent N,N'-dialyltartardiamide. The radioactivity can then be quantitated using liquid scintillation counting. The method is shown to be insensitive to reasonable variations in the intensity of staining as well as the gel volume over a practical range of gel slices. Recovery from the gel is extremely good with 93% of the counts associated with 14C-labeled proteins of known radioactive concentration being recovered. Analysis of a complex mixture of 3H-labeled proteins indicates resolution similar to that obtainable by autoradiography without the problems associated with quenching of autoradiographic signal by the staining procedure. The method is used to determine the amount of fucose and mannose incorporated into a putative cell adhesion protein during development of the cellular slime mold Dictyostelium purpureum.     

Automation of the computer handling of gel reading data produced by the shotgun method of DNA sequencing.

This paper describes a computer method for handling gel reading data produced by the shotgun method of DNA sequencing. The method greatly reduces the time the sequencer needs to spend checking and editing his data and yet it produces a consensus sequence for which the accuracy of determination of every base can be clearly shown. The program can take a batch of new gel readings, screen them against vector sequences removing any that match, and then compare and align all the sequences to produce a final consensus. No information is lost in this process as alignments are achieved by making only insertions and because all the individual gel readings are added to a database from which they can be retrieved and displayed lined up one above the other. This allows the user to check on the alignments achieved by the program and if necessary change them. As each gel reading is added to the database the consensus is automatically updated accordingly and used for the next comparisons. This is a much faster process than comparing each new gel against every individual gel in the database.     

A large-scale preparative electrophoretic method for the purification of pyridine nucleotide-linked dehydrogenases.

A large-scale preparative polyacrylamide gel electrophoresis (PAGE) method that uses a 1.5- or a 2.0-cm-thick slab gel has been developed for the purification of NAD-dependent dehydrogenases. With the 2.0-cm-thick gel, a maximum volume (up to about 160 ml) of enzyme sample was applied to a gel plate, resulting in the application of a large amount of protein and enzyme. After the electrophoretic run, the enzyme band on the gel was detected by activity staining and recovered from the gel by extraction with a fairly loose-fitting glass-Teflon homogenizer. NAD-dependent alanine dehydrogenase, leucine dehydrogenase, and glycerol dehydrogenase were purified in high yields (more than 80%) by the preparative PAGE method. The method can be carried out using a simple slab gel apparatus, which is modified from the conventional analytical apparatus for the purpose of preparative PAGE under conditions used for routine analytical runs. Thus, the method may be suitable for use in purifying NAD(P)-dependent dehydrogenases and many other enzymes after conventional chromatography such as dye-ligand affinity chromatography or ion-exchange chromatography.     

Quantitative assay of deoxyribonuclease activity after isoelectric focusing in polyacrylamide gels: pH control and effects of enzyme diffusion

A method for the quantitative assay of nuclease activity in crude cell lysates after isoelectric focusing (IEF) in polyacrylamide slab gels is described. After IEF, an agarose overlay gel containing DNA is placed on the IEF gel and the nuclease activity quantified by the loss of ethidium bromide fluorescence of the DNA. With this method a linear response was obtained for 1 to 10 ng of DNase I. Various methods of pH equilibration after IEF were also evaluated. The use of a high buffer concentration in the overlay gel is recommended to control the pH during the enzyme reaction. An analytical solution for the diffusion of enzymes from the IEF gel to the overlay gel is also presented and an equation that may be used to choose optimum times for transfer of the enzyme from the IEF gel to the overlay gel is given.     

One-step casting of Laemmli discontinued sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel

A modified Laemmli sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) protocol is described. The new method saves 30 min for gel casting without loss of the resolution power of Laemmli gel. In this method, both the upper and lower gels can be cast at the same time because the lower gel contains 10% glycerol, which generates higher density in the lower gel than in the upper gel.     

凝胶法生长溶菌酶晶体的研究

凝胶介质可以排除或削弱晶体生长过程中重力引起的对流和沉淀现象,用凝胶法生长生物大分子晶体是一种新的探索。使用类似于悬滴汽相扩散的方法,凝胶中生长出单个较大的外形发育完善且高度对称的鸡蛋清溶菌酶晶体。ＭＰＤ在凝胶中对溶菌酶结晶与溶液中具有相似的抑核作用。排循照像实验表明,凝胶法生长的晶体具有较强的衍射能力。     

A comparison between starch and polyacrylamide gels for the analysis of Listeria monocytogenes using multilocus enzyme electrophoresis

S.H. FLINT, N.J. HARTLEY, S.M. AVERY AND J.A. HUDSON. 1996. Forty-seven Listeria monocytogenes isolates were analysed using multilocus enzyme electrophoresis in two laboratories. Both assayed for the same six enzymes, but one used a starch gel method and the other polyacrylamide gels. The starch gel method distinguished six electrophoretic types whereas the polyacrylamide gel method produced 17 different electrophoretic types. The polyacrylamide gel method was more discriminatory than the starch gel method.     

Integration of multiple PCR amplifications and DNA mutation analyses by using oligonucleotide microchip

We have developed a method for parallel independent on-chip amplification and the following sequence variation analysis of multiple DNA regions directly using microchip with an array of nanoliter gel pads containing specific sets of tethered primers. The method has three key features. First, DNA to be amplified is enriched at gel pads by its hybridization with immobilized primers. Second, different sets of specific primers are immobilized within various gel pads, and primers are detached within gel pads just before polymerase chain reaction to enhance the amplification. A gel pad may contain an additional permanently immobilized dormant primer that is activated to carry out the allele-specific primer extension reaction to detect mutations. Third, multiple polymerase chain reactions are confined within nanoliter gel pads covered and separated from each other with mineral oil. The method was applied to simultaneously identify several abundant drug-resistant mutations in three genes of Mycobacterium tuberculosis.     

Oxygen diffusivity in gel beads containing viable cells

This article proposes a simple steady-state method for measuring the effective diffusion coefficient of oxygen (D(e)) in gel beads entrapping viable cells. We applied this method to the measurement of D(e) in Ca- and Ba-alginate gel beads entrapping Saccharomyces cerevisiae and Pseudomonas ovalis. The diffusivity of oxygen through gel beads containing viable cells was measured within an accuracy of +/-7% and found not to be influenced by cell density (0-30 g/L gel), cell type, and cell viability in gel beads. The oxygen diffusivity in the Ca-alginate gel beads was superior to that of the Ba-alginate gel beads, and the D(e) in the Ca-alginate gel beads nearly equalled the molecular diffusion coefficient in the liquid containing the gel beads. The oxygen concentration profile in a single Ca-alginate gel bead was calculated and compared to the distribution of mycelia of Aspergillus awamori grown in that gel bead. This procedure indicated that the oxygen concentration profile is useful for the estimation of the thickness of the cell layer in a gel bead. Numerical investigation revealed that high effectiveness factors, greater than 0.8, could be obtained using microgel beads with a radius of 0.25 mm.     

Protein patterns obtained from support-bound gels by combining the images at different stages of destaining

A more informative method for the visualization of proteins on thin-layer gels, based on combining the images of the gel at different stages of destaining, is presented. It is useful whenever important information seems to be lost after prolonged destaining. The method, which makes it unnecessary to run different loads in different channels, has been developed utilizing isoelectric focusing on polyester film-bound agarose gels. The strongly destained gel is superimposed on a negative image of the same gel made at an earlier phase of destaining, thus showing white spots on a gray background for minor components and dark bands in a white field surrounded by the grey background for the abundant ones. In general, the method may be applied to gel images obtained by different staining procedures.     

Segregation of partly melted DNA molecules

Segregation of partly melted DNA molecules is a convenient and efficient method to isolate DNA fragments associated with CpG islands. The method stands on the observation that the electrophoretic mobility of partly melted DNA fragments in a denaturing gradient gel is low and that they persist in the gel so long as the remaining helical part is sufficiently resistant to strand dissociation and dissociates slowly. Such features are observed in DNA fragments derived from CpG islands. These DNA fragments are preferentially retained in a denaturing gradient gel after prolonged electric field exposure, permitting the enrichment of DNA fragments derived from CpG islands. The principle and practical application of this method are reviewed.     

Detection of proteases by clotting of casein after gel electrophoresis

Clotting of casein provides a sensitive method for detection of proteases after gel electrophoresis. The method is here designated "caseogram." After electrophoresis the gel was equilibrated with 0.15-0.3 M sodium acetate, pH 5.3, and an 1% agarose gel containing 1% skim-milk powder in 0.1 M sodium acetate, pH 5.3, was placed on top of the electrophoresis gel. By incubation at 37 degrees C for 2 h the protease-containing zones produced distinct precipitates in the skim-milk gel. For permanent documentation the skim-milk gel was stained with amido black. The detection limit for pepsin A is 5 ng in the caseogram against 25 ng by hemoglobin digestion at pH 2.5. For calf chymosin it is 1 ng against 100 ng by digestion of hemoglobin at pH 3.5. Caseograms work well after agar gel electrophoresis, after different types of immunoelectrophoresis, and after isoelectric focusing or disc electrophoresis in polyacrylamide gels. Since inert proteins do not interfere with the detection, the method is especially suitable for analysis of crude samples. Samples containing pepsinogen or pepsinogen-like zymogens may be activated at pH 2 before equilibration at pH 5.3.     

Analysis of mitogenic activity of proteins after separation by gel electrophoresis

We have used a combination of gel electrophoresis and a cell culture assay in microplates to analyse mitogenic activity in tissue extracts. The procedure is a modification of the method described by Kuo et al. The proteins were separated by native gel electrophoresis or isoelectric focusing. The gel was sliced and defined pieces were transferred into tissue culture inserts fitting in 96 well microplates, which contained the test cells. The proteins diffused from the gel slices directly into the culture supernatant and the mitogenic effects were evaluated by a colorimetric assay (MTT or phosphatase activity). Human interleukin 2 was used to demonstrate the feasibility of the method by evaluating the mitogenic effect on the cell line CTLL-2. Extracts of bovine pituitary glands were separated by native gel electrophoresis and isoelectric focusing and several protein bands could be identified which showed a distinct mitogenic effect on human endothelial cells. The method is very sensitive and allows rapid screening of protein mixtures for bioactive fractions. This revised version was published online in July 2006 with corrections to the Cover Date.     

一种用于PCR模板制备的电泳产物简易回收方法

为了探索一种简便、有效而且能从琼脂糖凝胶中大量回收用于第2次PCR扩增的DNA电泳条带的方法,采用刀片切胶法和牙签插胶法从琼脂糖中回收DNA,并进行了两种方法的比较．结果显示牙签插胶法回收的DNA用作第2次PCR的模板,获得了清晰、稳定的PCR产物电泳条带,用该法成功地制备了一批DNA微阵列探针．由此可见牙签插胶法是一种简便、快速、有效的用于PCR模板的DNA琼脂糖凝胶回收法．