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.     

A versatile technique for drying polyacrylamide slab gels

Slab gels of different thickness, and of various homogeneous or gradient concentrations of acrylamide, can be dried rapidly and consistently using a newly developed drying procedure. After drying, stained gels retain excellent resolution of electrophoretic patterns, and unstained gels are suitable for use in autoradiography and fluorography.     

An improved method for arylsulfatase A detection on polyacrylamide slab gels.

An improved method for the detection of arylsulfatase A on polyacrylamide slab gels has been developed using a four-step reaction sequence which leads to the formation of a stable, localized, visible product. The reaction sequence involves the formation of nitrocatechol which, in turn, reduces cupric ferricyanide to form Hatchett's brown, an insoluble brown compound. The next steps involve polymerization of 3,3′-diaminobenzidine and osmication of the polymer. Since the initial reaction product, nitrocatechol, reacts quickly with the cupric ferricyanide to form the Hatchett's brown precipitate, little diffusion occurs and the observed bands of Hatchett's brown are highly localized. The formation of the Hatchett's brown can be observed as the arylsulfatase A reaction proceeds. The final osmium-stained product is very stable and has been stored for weeks without appreciable loss of light absorption or increase in band width. Analysis of arylsulfatase A activity in human leukocyte lysates after discontinuous electrophoresis in polyacrylamide slab gels demonstrated the presence of four bands of activity.     

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.     

Anomalous electrophoresis of deoxyribonucleic acid restriction fragments on polyacrylamide gels

A detailed study has been made of the polyacrylamide gel electrophoresis of DNA restriction fragments obtained from two plasmids, pBR322 and p82-6B. Variables studied were molecular weight, gel concentration, temperature, and electric field strength. The retardation coefficients of the larger fragments (greater than 800 base pairs) were independent of molecular weight. The retardation coefficients of the smallest fragments (less than or equal to 300 base pairs) were proportional to Mr1/3, and therefore to the mean geometric radii of the fragments. The logarithm of the relative mobility of all fragments was also proportional to Mr1/3. The anomalous migration of certain fragments on polyacrylamide gels was found to be "transportable" into fragments generated by different restriction enzymes. Anomalous migration was enhanced at lower temperatures and disappeared upon increasing the temperature. A fragment which migrated anomalously slowly migrated even more anomalously when dimerized; dimerizing a normally migrating fragment resulted in the normal migration of the dimerized fragment. Anomalously migrating fragments were found to be localized in distinct regions of the pBR322 circle.     

A visible dye-based staining method for DNA in polyacrylamide gels by ethyl violet

We describe a visible dye-based staining method for DNA in polyacrylamide gels using ethyl violet (EV). The novel method is a background-free, sensitive, economical, and simple procedure involving only staining and washing steps that can be completed within 30 min. As little as 0.8-1.6 ng of φX174 DNA/HaeIII can be detected by EV, which is about eightfold more sensitive than Nile blue (NB) stain and twofold less sensitive than ethidium bromide (EB) stain.     

A new method of sample application for horizontal slab polyacrylamide gel electrophoresis

A new method of sample application for horizontal slab polyacrylamide gel electrophoresis has been developed which solves the main problems associated with existing systems. A quick, simple procedure is described for placing a dry powder mixture of Celite and Sephadex into sample wells of any shape cut to the full depth of the gel slab. Samples can then be added to the powder to form a moist firm bed of material in the wells which prevents leakage of sample from the well. The method enables the quantitative electrophoresis of many samples with widely differing concentrations and volumes without the problems of electrodecantation, loss of electrical contact through the wells, or uneven penetration of sample through the full thickness of the gel.     

A discontinuous electrophoretic system for separating peptides on polyacrylamide gels

An electrophoretic system for separating, with high resolution, peptides 25–250 residues in length is described. The peptides are stacked by discontinuous electrophoresis to form very sharp bands at the origin. They are then separated on a matrix of 20% polyacrylamide, 8 m urea, and 0.1% dodecyl sulfate. Through this combination, high resolution and clean separation, based on polymer length, are achieved.     

A novel procedure for separating small peptides on polyacrylamide gels

A simple and fast procedure that allows the separation of small(1–3 kDa) peptides on glycine-SDS gels is described. Peptideswere separated by glycine-SDS/PAGE as a result of in situ complexation of peptide/SDS during electrophoretic migration and visualized by Coomassie blue staining. The data presented here shows the separation of small peptides of different isoelectric points, sizes, and hydrophobicity on polyacrylamidemini gels. Ten different peptides have been tested with this method. The data suggest the dependence of SDS/peptide complex formation and migration due to the number of basic amino acid residues, length of peptide and the hydrophobicity/hydrophilicity ratio.     

A novel procedure for separating small peptides on polyacrylamide gels

Summary A simple and fast procedure that allows the separation of small (1–3 kDa) peptides on glycine-SDS gels is described. Peptides were separated by glycine-SDS/PAGE as a result ofin situ complexation of peptide/SDS during electrophoretic migration and visualized by Coomassie blue staining. The data presented here shows the separation of small peptides of different isoelectric points, sizes, and hydrophobicity on polyacrylamide mini gels. Ten different peptides have been tested with this method. The data suggest the dependence of SDS/peptide complex formation and migration due to the number of basic amino acid residues, length of peptide and the hydrophobicity/hydrophilicity ratio.     

盘状聚丙烯酰胺凝胶电泳法分离血清蛋白实验的几点改进

对盘状聚丙烯酰胺凝胶电泳法分离血清蛋白实验提出了几点改进,以满足本科生实验的要求。实验主要比较和分析了两种封胶方法（原胶布封胶与改进的琼脂糖封胶）和两种染色方法（原考马斯亮蓝染色法与改进的考马斯亮蓝染色法）对凝胶分离血清蛋白实验的影响。结果显示,改进的盘状聚丙烯酰胺凝胶电泳法是一种灵敏、快速、简便、安全、分辨率高的实验方法。结论：改进的盘状聚丙烯酰胺凝胶电泳法分离血清蛋白实验非常适合本科生实验。     

低背景、高分辨率PAGE简易银染法

聚丙烯酰胺凝胶电泳银染一直存在耗时长、步骤繁琐等缺陷, 是其批量应用的瓶颈。文章报道了一种低背景、高分辨率的PAGE简易银染方法, 该法在降低NaOH浓度和批量显带方面进行了有益的探索, 建立了节省时间、节约试材, 对批量显带尤为实用的简易银染法。     

A new method for HDL particle sizing by polyacrylamide gradient gel electrophoresis using whole plasma

Low plasma levels of HDL cholesterol have been associated with an increased risk of coronary heart disease. HDL particles are heterogeneous with respect to size and apolipoprotein content. The objective of the present study was to develop a method to generate lipid-stainable calibrators that would allow the assessment of HDL particle size from whole plasma, using polyacrylamide gradient gel electrophoresis (PAGGE). Lipid-stainable HDL calibrators were obtained by subjecting isolated red blood cells to hemolysis either by freezing at -20 or -80 degrees C overnight or by rapid exposure to liquid nitrogen and mixing of the hemolysis products with plasma aliquots. All three methods were highly reproducible in producing Sudan black lipid-stainable HDL calibrators ranging from 75 to 200 A. The assessment of HDL particle size with these lipid-stainable HDL calibrators was also highly reproducible, with a coefficient of variation below 5.5%. These lipid-stainable HDL calibrators simplify the assessment of HDL particle size by PAGGE using whole plasma, without the need for costly, time-consuming ultracentrifugation procedures.     

A rotating temperature-controlled water bath for isozyme development in polyacrylamide slab gels

Plans are presented for an inexpensive heating bath which slowly rotates polyacrylamide or starch gels so that they are gently and continuously agitated in the dark. Rotation of the gels allows the use of significantly reduced amounts of biochemicals for isozyme development.