A method for silver staining HMG chromosomal proteins in polyacrylamide electrophoretic gels

A method is presented for sensitive staining of the HMG14 and 17 proteins in polyacrylamide gels pre-stained with Coomassie Blue R250. The procedure involves binding negatively and positively charged polycyclic aromatic compounds to the proteins followed by staining with silver using the method of Wray et al. (1981).     

Two-dimensional peptide mapping by polyacrylamide-gel electrophoresis with limited proteolysis in SDS

The peptide mapping method described by Cleveland, et al. (1) was improved to a two-dimensional analysis applicable to minute amounts (less than 0.5 microgram) of proteins. Radioiodinated proteins for analysis were purified by electrophoretic elution of the proteins from polyacrylamide gels into buffer containing 0.1% sodium dodecyl sulfate. The proteins were digested enzymatically in the presence of 0.1% sodium dodecyl sulfate and an excess of nonlabeled bovine serum albumin (0.2 mg/ml) relative to labeled substrate in order to attain reproducibility by maintaining a consistent substrate concentration among different samples. The peptides of these limited proteolytic products were resolved by two-dimensional polyacrylamide gel electrophoresis (isoelectric focusing followed by SDS-gels). The resulting 2D-peptide maps of murine and bovine albumin and a murine lymphocyte membrane protein, Tp100, showed excellent resolution and reproducibility.     

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.     

An improved method of sequential alcian blue and ammoniacal silver staining of chondroitin sulfate proteoglycan in polyacrylamide gels

Partially deglycosylated chondroitin sulfate proteoglycan (CSPG) or peptide fragments obtained from CSPG are not readily detectable in gels by staining with Alcian blue 8GX or ammoniacal silver using the technique of Oakley et al. (B. Oakley, D. Kirsh, and N. Morris (1980) Anal. Biochem. 105, 361). Sequencial staining with both reagents allows visualization of intact CSPG or peptides derived from proteoglycans in polyacrylamide gels at protein concentrations as low as 2 ng/mm2, or glucuronic acid and galactosamine concentrations of 1 ng/mm2 or less. This method is significantly more sensitive and has broader applicability than that described by H. Min and M. Cowman (1986) Anal. Biochem. 155, 275) for staining glycosaminoglycan fragments in polyacrylamide gels.     

Dephosphorylation of histones H1 and H3 during the isolation of metaphase chromosomes.

Histones have been extracted from isolated metaphase chromosomes prepared by the method of Wray and Sutbblefield [Exp. Cell Res 59, 469-478 (1970)] and by a Nonidet P-40 detergent procedure based on the method of Wigler and Axel [Nucleic Acids Res. 3, 1463-1471 (1976)]. Analysis of the densitometer profiles of long polyacrylamide gels shows that the mitotic phosphorylations of histone H1 (H1M) and histone H3 are extensively depleted during chromosome isolation. These data indicate that CHO metaphase chromosomes prepared by standard methodologies do not represent in vivo chromosomes with respect to their histone phosphorylations; therefore, current chemical and structural studies of isolated metaphase chromosomes may require further clarification.     

Quantitation of specific proteins in polyacrylamide gels by the elution of fast green FCF

The quantitation of proteins in polyacrylamide gels stained with Fast Green FCF has been investigated using a modification of the elution technique originally described by Fenner et al. (Fenner, C., Traut, R.R., Mason, D.T. and Wikman-Coffelt, J. (1975) Anal. Biochem. 63, 595–602) for Coomassie Blue and adapted by Medugorac (Medugorac, I. (1979) Basic Res. Cardiol. 74, 406–416) for use with proteins stained with Fast Green FCF. The elution of dye from stained protein was accomplished using 1.0 M NaOH instead of aquoeus pyridine as required by the original method. The primary advantages of our modification are that the time required for protein quantitation has been considerably reduced and the use of toxic organic solvents has been eliminated. We have investigated the applicability of the method to several different proteins and our results indicate: (a) The quantity of Fast Green eluted from specific proteins is proportional to the quantity of protein applied to the gel, but varies for each individual protein. (b) The method allows quantitation over a very wide range of protein (1–800 μg). (c) Quantitation of protein is independent of the width of the stained bands as well as acrylamide concentration. (d) The method is applicable to gels of many types including disc, slab and continuous gradient gel, (e) Protein can be estimated from the patterns obtained by two-dimensional polyacrylamide gel electrophoresis. (f) The presence of Triton X-100 in gel and protein sample does not affect quantitation; the method is applicable to gels containing SDS provided that SDS is removed prior to staining. (g) Precipitation of protein with 12.5% TCA following electrophoresis does not interfere with quantitation. (h) The reproducibility of the technique is excellent, with standard deviations being less than 10% of the mean in all cases. This method appears highly versatile but requires appropriate standards for the quantitation of individual proteins.     

Detection of subnanogram amounts of RNA in polyacrylamide gels in the presence and absence of protein by staining with silver

The new ultrasensitive photochemically derived silver stain described for polypeptides in polyacrylamide gels (Merril et al., Science211, 1437–1438 (1981)) also stains nucleic acid in polyacrylamide gels. Reovirus genome double-stranded (ds) RNA segments were clearly detected in gels at about 0.03 ng/mm² with the silver staining technique when either purified virions or isolated, purified dsRNA was analyzed. The silver stain was about 10 to 30 times more sensitive than ethidium bromide for detecting reovirus dsRNA.     

Nuclear ribonucleoprotein complexes containing U1 and U2 RNA.

Nuclear ribonucleoprotein (RNP) complexes that contain the U1 and U2 RNA of chromatin of Novikoff hepatoma cells were extracted with 0.01 M Tris-HCl (pH 8.0) after the nuclei were initially washed with 0.075 M NaCl and 0.025 M EDTA (pH 8.0). These RNP complexes were purified by chromatography on Sepharose 6B columns and centrifugation on sucrose density gradients. The identity of the U1 and U2 RNA in these particles was established by their electrophoretic mobility in polyacrylamide gels and their T1 RNase fingerprints which were identical with those of authentic U1 and U2 RNA (R. Reddy et al. (1974), J. Biol. Chem.249, 6486-6494; H. Shibata et al. (1974), Mol. Cell. Biochem. 4, 3-19). The nuclear riboncleoproteins had a buoyant density of 1.47 g/ml in CsCl gradients. Two-dimensional polyacrylamide gel electrophoresis of their proteins showed these RNP complexes contain 10 polypeptide spots, of which two are phosphorylated in vivo.     

Characterization of histones from beef pancreas]

The total histone of ox pancreas was fractionated by electrophoresis on 10 and 25 cm polyacrylamide gels according to Panyim and Chalkley (1969). The presence of an additional subfraction within the lysine rich histone was stated. In the course of the fractionation of total histone according to the method of Oliver et al. (1972) this additional histone component was extracted together with F1 histone.     

Influence of superinfection on the photoreversible phase of UV induced lysogenic bacteria

Summary Bombyx mori L. ribosomal proteins have been analyzed by four related two-dimensional polyacrylamide gel electrophoretic systems (Madjar et al. 1977a). In the small and large subunits are present 32 and 45 proteins, respectively, whose numbering is proposed. No significant differences in composition or migration could be detected between proteins in membranebound ribosomes and free ribosomes. The molecular weights of the proteins vary from 60,000 to less than 10,000. In vivo phosphorylation was investigated by labeling with ³²P-orthophosphate. Autoradiograms of four two dimensional gels unambiguously show five labeled ribosomal proteins: S1, S7, L6, L29, and L40.     

The tritium labeling of small amounts of protein for analysis by electrophoresis on sodium dodecyl sulfate--polyacrylamide slab gels.

A simple and reproducible method for the tritium labeling of small amounts of proteins prior to analysis under denaturing conditions on polyacrylamide slab gels is described. The method involves the in vitro labeling of proteins by reductive methylation using formaldehyde and high specific activity [³H]potassium borohydride. Labeled proteins were detected by fluorography after fractionation on polyacrylamide slab gels in the presence of sodium dodecylsulfate. The overall procedure allows the analysis and molecular weight estimation of submicrogram quantities of protein.     

Electrophoretic elution of proteins from polyacrylamide gel slices

A method for electrophoretic elution of proteins from polyacrylamide gel slices is described. Eluted proteins were retained by a discontinuous conductivity gradient (M. Otto and M. Snejdárková, Anal. Biochem. 111, 111-114 (1981)). The method has been adapted to slices from slab gels and gels that have been stained and destained. Proteins were eluted as their sodium dodecyl sulfate complexes. Minute amounts of proteins (0.1 microgram) were recovered in high yield (85-95%) in 2 h in less than 0.1 ml volume.     

Relation Between the Chemical Structure and the Biological Activity of Hydroxystilbenes Against Botrytis cinerea

Abstract A Spanish strain of pepper mild mottle virus (PMMV-S) (A lonso et al. 1989) can be differentiated from the Italian PMMV (W etter et al. 1984) by the responses of Capsicum spp. with resistance genes to tobamoviruses, by radioimmunoassay and by the electrophoretic mobility of their viral particles. Moreover, the analysis of the electrophoretic mobility of the viral particles in agarose gels and that of the viral coat proteins in polyacrylamide-urea gels are reliable and rapid techniques for distinguishing PMMV isolates from other members of the tobamovirus group and thus can be used for diagnostic purposes. These results support the proposal of grouping these pepper viruses, including the "pepper strains" of TMV, as a new tobamovirus subgroup (PARES 1985, BETTI et al. 1987, WETTER et al. 1987, ALONSO et al. 1989).     

SUBCELLULAR FRACTIONATION STUDIES RELATED TO THE PROCESSING OF NEUROSECRETORY PROTEINS IN APLYSIA NEURONS

Abstract— Neurosecretory cells (bag cells and R_3–14 neurons) in the abdominal ganglion of Aplysia californica were 'pulse-chased' in [³H]leucine and comparisons of the labeled protein profiles from the total cell homogenate versus a crude 'neurosecretory granule' fraction on acid-urea polyacrylamide gels were made, The data provides indirect support for the hypothesis that some of the post-translational processing of the neurosecretory proteins occurs intragranularly (L oh et al , 1975). In the case of the Bag cells the initial processing of the 29,000 daltons precursor appears to occur extragranularly, possibly in the rough endoplasmic reticulum cisternae.     

Preparative scale, high resolution purification of low molecular weight DNA fragments

A commercially available continuous electroelution system has been used to separate and purify low molecular weight DNA fragments from polyacrylamide gels. This method has several advantages over previously reported methods for the recovery of DNA fragments from polyacrylamide gels. This technique, which gives very high recovery rates (80-95%), can be carried out on a relatively large scale and in a way that is not labour intensive. Data are presented for the purification of DNA fragments with molecular weights in the range 1-4 x 10(5) (200-700 base-pairs), although the method is also applicable to larger molecular weight DNA fragments, RNAs and proteins.     

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.     

Isolation of proteins for peptide mapping, amino acid analyses, and sequencing using disulfide crosslinked polyacrylamide gels

Conditions for recovery of small amounts of proteins (1-50 micrograms) from disulfide crosslinked polyacrylamide gels have been examined. Procedures were developed for solubilization and precipitation of Coomassie blue-stained protein bands excised from gels after electrophoretic separations. The precipitated protein was then resolubilized for use in peptide mapping, amino acid analyses, or microsequencing. The amino acid compositions of standard proteins (bovine albumin, ovalbumin, phosphorylase b, and beta-galactosidase) isolated by this method were in good agreement with the values for the corresponding conventionally purified proteins. Sequencing was done with high repetitive yield on samples of 100 pmol or below. The method has been successfully applied to several proteins and protein fragments.     

Improved Ruthenium II tris (bathophenantroline disulfonate) staining and destaining protocol for a better signal-to-background ratio and improved baseline resolution

In proteomics the ability to visualize proteins from electropherograms is essential. Here a new protocol for staining and destaining gels treated with Ruthenium II tris (bathophenantroline disulfonate) is presented. The method is compared with the silver-staining procedure of Swain and Ross, the Ruthenium II tris (bathophenantroline disulfonate) stain described by Rabilloud (Rabilloud T., Strub, S. M. Luche, S., Girardet, S. L. et al., Proteomics 2001, 1, 699-704) and the SYPRO Ruby gel stain. The method offers a better signal-to-background ratio with improved baseline resolution for both sodium dodecyl sulfate-polyacrylamide gels and two-dimensional gels.     

Fast and sensitive silver staining of DNA in polyacrylamide gels.

The photochemically derived silver stain of nucleic acids in polyacrylamide gels originally described by Merril et al. (1981, Science 211, 1437-1438) was modified to reduce unspecific background staining and increase sensitivity (down to 1 pg/mm2 band cross-section). Detection limits for double-stranded DNA fragments from HaeIII endonuclease digests of phage phi X174 were maintained despite eliminating oxidation pretreatment of fixed gels and reducing silver nitrate concentration. Preexposure to formaldehyde during silver impregnation enhanced sensitivity and the inclusion of the silver-complexing agent sodium thiosulphate in the image developer decreased background staining. Higher formaldehyde concentration during image development resulted in darker bands with good contrast. The procedure almost halves the number of steps, solutions and experimental time required and can be used for the staining of DNA fragments in polyacrylamide gels bound to a polyester backing film by controlling temperature during image development. We have applied this improved staining procedure for the routine analysis of complex DNA profiles generated by DNA amplification fingerprinting (DAF).