Visible fluorescent detection of proteins in polyacrylamide gels without staining

2,2,2-Trichloroethanol (TCE) incorporated into polyacrylamide gels before polymerization provides fluorescent visible detection of proteins in less than 5min of total processing time. The tryptophans in proteins undergo an ultraviolet light-induced reaction with trihalocompounds to produce fluorescence in the visible range so that the protein bands can be visualized on a 300-nm transilluminator. In a previous study trichloroacetic acid or chloroform was used to stain polyacrylamide gel electrophoresis (PAGE) gels for protein visualization. This study shows that placing TCE in the gel before electrophoresis can eliminate the staining step. The gel is removed from the electrophoresis apparatus and placed on a transilluminator and then the protein bands develop their fluorescence in less than 5min. In addition to being rapid this visualization method provides detection of 0.2microg of typical globular proteins, which for some proteins is slightly more sensitive than the standard Coomassie brilliant blue (CBB) method. Integral membrane proteins, which do not stain well with CBB, are visualized well with the TCE in-gel method. After TCE in-gel visualization the same gel can then be CBB stained, allowing for complementary detection of proteins. In addition, visualization with TCE in the gel is compatible with two-dimensional PAGE, native PAGE, Western blotting, and autoradiography.     

A silver staining procedure for nucleic acids in polyacrylamide gels without fixation and pretreatment

Silver staining of nucleic acid has been widely used in molecular marker analysis such as simple sequence repeat (SSR), single-strand conformation polymorphism (SSCP), and amplified fragment-length polymorphism (AFLP). Many alternatives to silver staining methods have been described, but these methods are not efficient or cost-effective. Here we report a silver staining method that requires less than 10 min for one gel and can save chemicals as well. It has a detection limit of approximately 5.6 pg of DNA/mm² in nondenaturing polyacrylamide gels and 12.8 pg/mm² in denaturing polyacrylamide gels.     

Chromium (III) enhanced diamine silver staining of proteins and DNA in gels

Chromium (III) enhanced the sensitivities of diamine silver staining of four proteins between 6- and 50-fold over that of the Coomassie Brilliant Blue (CBB)-chromium modified thiosulfate-silver staining method (Zhou et al. Biotechnology Letters, 2002, 24: 1561–1567). Using six dsDNA fragments, the detection limits of this new method was 10 to 30 pg per band, being 10- to 25-fold more sensitive than previous methods.     

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.     

Detection of dextransucrase and levansucrase on polyacrylamide gels by the periodic acid-Schiff stain: staining artifacts and their prevention

One use of the periodic acid-Schiff (PAS) stain is to detect dextransucrase and levansucrase activities on polyacrylamide gels by staining their polysaccharide products, dextran and levan. When gels with heavy dextran or levan bands were PAS stained, proteins other than dextransucrase and levansucrase also were stained, and a high background developed during storage. The staining of proteins other than dextransucrase and levansucrase is caused by the diffusion of the periodate-oxidized carbohydrate before and after staining. This diffusion could be greatly slowed, and the staining artifact decreased, by following the PAS stain by a crosslinking treatment of the carbohydrate-dye complex. Protein staining artifacts could be prevented by using chymotrypsin to remove the protein from the gel at the stage after polysaccharide synthesis but before the PAS stain.     

Silver staining of proteins in polyacrylamide gels

An automatic method for the protein assay using Coomassie Brilliant Blue G-250 was developed and applied to the assay of urinary proteins. In developing the automatic system, the adhesion of protein-bound dye to the walls of the flow cell and tubes was found to be the most troublesome problem, by which the baseline was shifted upwardly to give positive errors. For the purpose of preventing such adhesion, the concentration of CBB was reduced to half of that used in the manual method, glass tubes and glass coils were changed to those made of Kel-F material, and the flow cell was coated with fluorine resin. As a result, the staining with protein-bound dye was nearly completely eliminated. The final system showed satisfactory ability in performance, namely, the value of a coefficient variation for the reproducibility within run was 1.3%, that for the carry over was 0–1.1%, and the recovery was 98.8%. The calibration curve was linear in a range of 0–1000 μg/ml, and 80 samples could be processed in 1 h. Thus, the present method may serve as an efficient automatic protein analyzer for routine clinical tests of urine samples.     

Murine serum deoxyribonuclease 1 (Dnase1) activity partly originates from the liver

Reduction of serum DNASE1 (DNase I) activity is supposed to aggravate anti-nuclear autoimmunity, i.e. Systemic Lupus Erythematosus (SLE) in man and mice. To evaluate the etiology of this reduction, more information is needed about the source(s) and regulation of serum DNASE1. In this work we used male C57BL/6 wild-type (WT) mice to verify that serum Dnase1 activity partly depends on hepatic Dnase1 gene expression. Thus serum and liver Dnase1 activity showed a parallel oscillatory course during 24h, which was accompanied by a phase-shifted fluctuation of the hepatic Dnase1 mRNA content. Performing native PAGE zymography (NPZ) we detected a presumably premature non-sialylated and a mature sialylated hepatic Dnase1 isoform, which both show a parallel circadian fluctuation, indicating continuous secretion of Dnase1. The sialylated form was also detectable in serum. By immunostaining the hepatocytes were identified as the source of hepatic Dnase1 gene expression. After 70% hepatectomy, the serum Dnase1 activity increased markedly due to the occurrence of ischemic hepatocellular necrosis in the vicinity of the surgical suture. Similarly, hepatocellular necrosis induced by injection of streptolysin-O (SLO) into the liver led to a rapid parallel increase of Dnase1 and of aspartate- and alanine aminotransferase (AST/ALT) in serum. Subsequent to hepatectomy, Dnase1 gene expression was up-regulated in the regenerating liver most likely leading to an enhanced serum Dnase1 level until complete regeneration. These data demonstrate that serum Dnase1 at least partly originates from the liver and hint to the possibility that natural as well as pathological hepatic conditions influence its activity.     

Human serum deoxyribonuclease I (DNase I) polymorphism: pattern similarities among isozymes from serum, urine, kidney, liver, and pancreas.

We have devised a zymogram method with high sensitivity and resolution for investigating molecular heterogeneity and genetic polymorphism of deoxyribonuclease I. A combination technique of polyacrylamide-gel isoelectric-focusing electrophoresis and the newly developed zymogram method have led to the discovery of genetic polymorphism of human serum DNase I. Family studies showed that the three common phenotypes--DNASE1 1, DNASE1 1-2, and DNASE1 2--and the other five relatively rare phenotypes--DNASE1 1-3, DNASE1 2-3, DNASE1 2-4, and DNASE1 3-4--represent homozygosity or heterozygosity for four autosomal codominant alleles, DNASE1 *1, DNASE1 *2, DNASE1 *3, and DNASE1 *4. The frequencies of DNASE1 *1, DNASE1 *2, DNASE1 *3, and DNASE1 *4 calculated in a Japanese population were .5517, .4358, .0104, and .0021, respectively. Moreover, it was found that urine and extracts of kidney, liver, and pancreas, as well as serum, can be used for DNase I phenotyping.     

Silver staining for carboxymethylated metallothioneins in polyacrylamide gels

A sensitive method for detecting metallothioneins (MTs) by use of silver staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of carboxymethylated MTs was developed. Carboxymethylation of metallothioneins is indispensable because it prevents their aggregation, thereby allowing each of them to be detected as a single band by SDS-PAGE. However, when the gel was subjected to the silver-staining method of C. R. Merril, D. Goldman, S. A. Sedman, and M. H. Ebert [(1981) Science 211, 1437-1438], the image of carboxymethylated purified MTs was totally negative. Pretreatment of the gel with 1% sodium thiosulfate just prior to the silver-staining procedure successfully reversed the negative image of carboxymethylated MTs. Further, they could be detected with a limit of nanogram levels per lane. This method can be applied to MTs in cell extracts from cultured cell lines treated with cadmium or to those from liver of cadmium-intoxicated mice.     

Activity staining of endoglucanases in polyacrylamide gels.

The endoglucanases of Penicillium funiculosum were analyzed for the presence of multiple forms using a modified version of the Congo red method. Postelectrophoretic slab gels were directly incubated in a solution of carboxymethylcellulose for a period as short as 15 min and then the activities were visualized by staining with Congo red. Ten distinct bands of clearances were obtained indicating the presence of at least as many multiple forms.     

Use of methyl green-DNA agarose for detecting deoxyribonuclease activity in polyacrylamide gels

A liquid scintillation counting system is described that allows recovery of compounds for further study and analysis. For most classes of compounds tested (with the exception of steroids) the recovery was high (usually at least 90%) and in the case of nucleosides was accompanied by very little degradation of the sample. The counting method should be useful for the counting of samples where a high recovery of the compound is necessary.     

Paper chromatographic assay of ( 3 H)norepinephrine and its five major metabolites

The technique described here offers several advantages over the currently available assays for [³H]norepinephrine and its metabolites. The technique is simpler and has fewer sources of potential error than most currently available techniques. It provides a complete separation of norepinephrine from its five major metabolites with virtually no cross-contamination between the fractions. It is unnecessary to correct for recovery, elution from the chromatograms, or efficiency of counting the chromatogram segments. Experiments reported here demonstrate that all five major metabolites of norepinephrine are formed by rabbit aorta. Most of the neutral metabolites and normetanephrine are found in the Krebs solution used for incubating the tissue, whereas most of the acid metabolites are found in the tissue.     

Ion-enhanced fluorescence staining of sodium dodecyl sulfate-polyacrylamide gels using bis(8-p-toluidino-1-naphthalenesulfonate)

A method for the sensitive fluorescent staining of sodium dodecyl sulfate (SDS) gels that extends the applicability and sensitivity of existing procedures has been developed. SDS-protein complexes are able to bind the noncovalent hydrophobic probe, bis(8-p-toluidino-1-naphthalenesulfonate) (bisANS) with an increase in quantum yield that is considerably larger than that observed with the commonly used monomeric form, 1-anilinonaphthalene-8-sulfonic acid (1,8-ANS). The quantum yield of bisANS bound to the SDS-protein complex is greatly enhanced by incubation with one of a number of cations including potassium and barium. The use of bisANS with metal ion enhancements provides a method for staining SDS gels that can be more sensitive than commonly used methods based on the binding of Coomassie blue, and provides a simple and rapid method for the detection and quantitation of proteins. The use of metal ion enhancements also greatly increases the sensitivity of staining methods based on the use of 1,8-ANS. The present method is much more sensitive than previous noncovalent, flourescent, postelectrophoresis stains, but it retains their considerable advantages of speed, simplicity, and the ability to perform secondary procedures on the separated materials.     

Determination of poly(ADP-ribose) chain length distribution on polyacrylamide gels by silver staining.

Silver staining and polyacrylamide gel electrophoresis were used to visualize chain length distribution of poly(ADP-ribose) enzymatically synthesized from NAD by rat liver nuclei. The method described has the advantage that synthesis does not require radioactive-labeled NAD, and microgram quantities (greater than 5 micrograms) of poly(ADP-ribose) can be resolved and visualized as discrete bands according to chain lengths which range from 8 to 60 residues. This method can be applied to estimate size distribution of poly(ADP-ribose) chains in cells or tissues.     

Microbial methodological artifacts in [3H]glutamate receptor binding assays

Incubation of radiolabeled L-glutamic acid, a putative central excitatory neurotransmitter, in 50 mM Tris-acetate buffer (pH 7.4) at 30 degrees C in the absence of brain synaptic membranes resulted in a significant adsorption of the radioactivity to glass fiber filters routinely employed to trap the bound ligand in receptor binding assays. The adsorption was not only eliminated by the inclusion of L-isomers of structurally related amino acids, but also inhibited by that of most presumed agonists and antagonists for the brain glutamate receptors. This displaceable adsorption was a temperature-dependent nonreversible, and saturable phenomenon. Scatchard analysis of these data revealed that the adsorption consisted of a single component with an apparent dissociation constant of 73 nM. The displaceable adsorption was significantly attenuated by a concurrent incubation with papain, pronase E, and phospholipase C. A significant amount of the radioactivity was detected in the pass-through fraction of the Dowex column following an application of the reaction mixture incubated with purified [3H]glutamate at 30 degrees C for 60 min in the absence of membranous proteins added. Complete abolition of the displaceable adsorption resulted from the use of incubation buffer boiled at 100 degrees C as well as filtered through a nitrocellulose membrane filter with a pore size of 0.45 micron immediately before use. These results suggest that the displaceable adsorption may be attributable to the radioactive metabolite of [3H]glutamate by microorganisms contaminating the Tris-acetate buffer. This might in part contribute to some of the controversial results with regard to receptor binding studies on acidic amino acids.     

Eosin Y staining of proteins in polyacrylamide gels.

A staining method is described in which various proteins in polyacrylamide gels can be stained by using eosin Y. After a brief incubation of a polyacrylamide gel in an acidic solution of 1% eosin Y, various proteins, including human erythrocyte membrane sialoglycoproteins which are not detectable by Coomassie blue R-250 (CB), can be detected with a sensitivity of 10 ng protein. This is far more sensitive than CB staining and is comparable to the sensitivity of silver staining. In a Western blot, the antigenicity of an eosin Y stained protein is retained. In addition, proteins on an immunoblot sheet can be detected by eosin Y staining. The method described is rapid, sensitive, and reproducible with various proteins in polyacrylamide gels and has the added advantage of also staining sialoglycoproteins.     

Bioluminescent assay of D-3-hydroxybutyrate in serum

An enzymatic assay of D -3-hydroxybutyrate in which the hydroxybutyrate dehydrogenase reaction is coupled to the bacterial oxidoreductase—uciferase system is described. The bioluminescent assay is based on either, end-point, or on initial velocity measurements. This simple and rapid assay requires a single serum sample of 10 μl. Its linear range covers two orders of magnitude from 10^?6 mol/I upwards. This assay is suitable for the routine determination of D -3-hydroxybutyrate in human blood with good accuracy.     

Nonenzymatic exchange of tritium from (5-3H) deoxyuridylate in the thymidylate synthetase assay

When 2′-[5-³H] deoxyuridine-5′-phosphate and 2-mercaptoethanol are used to assay thymidylate synthetase activity, the tritium of 2′-[5-³H] deoxyuridine-5′-phosphate exchanged nonenzymatically with water. The rate of the isotope exchange depended on the concentrations both 2-m2-mercaptoethanol and amine buffer and the pH of the assay mixture. Such an exchange is a possible source of serious error in the assay when low activity of enzyme is assayed with highly radioactive 2′-deoxyuridine-5′-phosphate but can be minimized by maintaining a low concentration of 2-mercaptoethanol and unprotonated amine buffer in the assay mixture.