A simple and inexpensive procedure for preparative polyacrylamide gel electrophoresis of RNA

A method that is both simple and inexpensive is described for preparative polyacrylamide gel electrophoresis of low-molecular-weight RNA. The utility of the method is demonstrated by the preparative separation of 4S and 5S RNA.     

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 simple method for detection of neurosecretory products after polyacrylamide gel electrophoresis

Neurophysins, a family of cystine-rich proteins found in the hypothalamic-neurohypophysial system can be located histologically with aldehydefuchsin. A method is described whereby the aldehyde-fuchsin technique has been used to locate neurophysins after polyacrylamide gel electrophoresis.     

A simple method for isolating pure RNA preparations after electrophoresis in polyacrylamide gel

Upon gel electrophoresis under standard conditions, RNA from the Russian strain of barley stripe mosaic virus (BSMV) yielded two components. The gels were stained with ethidium bromide, and the RNA-containing zones were excised and extracted. The RNA isolated was precipitated from the dilute (8–10 μg/ml) extract by addition of 0.2 CaCl₂. The RNA preparation was, thereby, completely freed from polyacrylates present in the extract. The extracted components of the virus RNA had the same electrophoretic mobilities as those of the starting RNA preparation. They also retained their biological and messenger activity.     

A method for the long-term preservation of polyacrylamide gel electrophoresis

A new, reversible method for drying polyacrylamide gel electrophoresis is reported. It was studied using proteins from the B17, B20, B21 and ATCC 8014 strains of Lactobacillus plantarum isolated from the brine of table olives. After electrophoretic analysis, the gels were dehydrated in a 95% ethyl alcohol solution and stored either long-term or for a few days, renatured and then subjected to analyses that included combination staining with Coomassie brilliant blue and silver, and western blotting. The immunological tests and electrophoresis performed with the enzymes β-glucosidase, alkaline phosphatase and peroxidase demonstrated that repeated dehydration and renaturation of the polyacrylamide gels does not denature the proteins. The method is simple to perform, inexpensive and does not require special equipment.     

A method for staining and stabilizing peroxidase activity in polyacrylamide gel electrophoresis

A procedure was developed for a rapid double staining of peroxidase and other proteins in the same polyacrylamide gels using guaiacol and Coomassie blue. The distinguishable colored bands of peroxidase isozymes and proteins are stable for at least 8 months.     

A new convenient column for gel electrophoresis,isoelectric focusing,and zonal electrophoresis

A simple preparative electrophoresis column that can be utilized for gel and zonal electrophoresis and isoelectric focusing has been constructed.     

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 method for the direct measurement of glycogen synthase activity on gels after polyacrylamide gel electrophoresis

A method for the detection of glycogen synthase activity after nondenaturing polyacrylamide gel electrophoresis is described. After the electrophoretical run, the gels were incubated in situ with UDP-glucose and glycogen. Labeled or unlabeled UDP-glucose could be used, since similar activity patterns were obtained by autoradiography or iodine staining of the gels. The method here described offers several advantages in terms of speed, sensitivity, and economy when compared with other procedures.     

A method for in-gel fluorescent visualization of proteins after native and sodium dodecyl sulfate polyacrylamide gel electrophoresis

We have developed a simple one-step 30-min method for fluorescent visualization of proteins in native and sodium dodecyl sulfate polyacrylamide gel electrophoresis (PAGE) gels. The method is based on formation of strong fluorophores via potassium ferricyanide-provoked oxidation of tryptophan (Trp). Following PAGE, gels are soaked in water solution of potassium ferricyanide (100 mM) and NaOH (1 M) and are kept in the dark for 30 min. Gels are then transferred to water and scanned. The sensitivity of the method was slightly lower compared with standard Coomassie Brilliant Blue (CBB) staining. The method can be useful when rapid acquisition of data is of the essence. After preview, gels can be post-stained using the CBB protocol for further analysis. The intensity of fluorescence is dependent on Trp number, so the protocol might find application in the quantification of Trp residues as illustrated here. Importantly, there is room for improvement of the method. Namely, according to excitation–emission matrix analysis of stained protein bands, maximal fluorescence intensity (at 345/460 nm) was 3.5-fold higher compared with the settings that were available on a commercial imager (395/525 nm). As a supplement, we present an upgrade of the previously described method for in-gel detection of non-heme iron-binding proteins that also employs potassium ferricyanide.     

Detection of chitinase activity after polyacrylamide gel electrophoresis

Commercial Streptomyces griseus and Serratia marcescens chitinases and purified wheat germ W1A and hen egg white lysozymes were subjected to polyacrylamide gel electrophoresis under native conditions at pH 4.3. After electrophoresis, an overlay gel containing 0.01% (W/V) glycol chitin as substrate was incubated in contact with the separation gel. Lytic zones were revealed by uv illumination with a transilluminator after staining for 5 min with 0.01% (W/V) Calcofluor white M2R. As low as 500 ng of purified hen egg lysozyme could be detected after 1 h incubation at 37 degrees C. One band was observed with W1A lysozyme and several bands with the commercial microbial chitinases. The same system was also used with native polyacrylamide gel electrophoresis at pH 8.9. Several bands were detected with the microbial chitinases. The same enzymes were also subjected to denaturing polyacrylamide gel electrophoresis in gradient gels containing 0.01% (W/V) glycol chitin. After electrophoresis, enzymes were renatured in buffered 1% (V/V) purified Triton X-100. Lytic zones were revealed by uv after staining with Calcofluor white M2R as for native gels. The molecular weights of chitinolytic enzymes could thus be directly estimated. In denaturing gels, as low as 10 ng of purified hen egg white lysozyme could be detected after 2 h incubation at 37 degrees C. Estimated molecular weights of St. griseus and Se. marcescens were between 24,000 and 72,000 and between 40,500 and 73,000, respectively. Some microbial chitinases were only resistant to denaturation with sodium dodecyl sulfate while others were resistant to sodium dodecyl sulfate and beta-mercaptoethanol.     

A single nucleotide polymorphism genotyping method using phosphate-affinity polyacrylamide gel electrophoresis

To date, various methods have been developed to facilitate the genotyping of a single nucleotide polymorphism (SNP) for aiding in the diagnosis and treatment of inherited diseases. The most commonly used method for SNP genotyping is an allele-specific hybridization procedure using an expensive fluorochrome-labeled oligonucleotide probe and a specialized fluorescence analyzer. Here, we introduce a simple and reliable genotyping method using a 1:1 mixture of 5'-phosphate-labeled and nonlabeled allele-specific polymerase chain reaction (PCR) primers. The method is based on the difference in mobility of the phosphorylated and nonphosphorylated PCR products (in the same number of basepairs) on phosphate-affinity polyacrylamide gel electrophoresis. The phosphate-affinity site is a polyacrylamide-bound dinuclear zinc(II) complex, which preferentially captures the 5'-phosphate-labeled allele-specific product compared with the corresponding nonlabeled product. The obtained DNA migration bands can be visualized by ethidium bromide staining. We demonstrate the genotyping of a SNP reported in a human cardiac sodium channel gene, SCN5A, using this novel procedure.     

A high resolution PAS stain for polyacrylamide gel electrophoresis

An improved PAS method for the detection of glycoproteins after electrophoresis on acrylamide or urea-acrylamide gels is described. Stronger oxidizing conditions and a more controlled washing for the removal of periodic acid resulted in both increased staining intensity and band resolution. The method will not stain protein and it appears that a 2–3 μg sample of bound or precipitable carbohydrate would easily be detected. Mucopolysaccharides were not detectable by this method.