Silver-stained Polyacrylamide Gels Research Articles

MOLECULAR CLONING OF DSRNAS ASSOCIATED WITH STRAWBERRY MOTTLE VIRUS

Strawberry mottle virus was transmitted from Fragaria vesca to Chenopodium quinoa and Nicotiana occidentalis 37B. In all plants, SMoV infection-associated dsRNA was detected but dsRNA patterns showed some differences between the herbaceous hosts. The differences were more evident when the dsRNA preparations were electrophoresed employing a SDS-PAGE method followed by silver staining of polyacrylamide gels than when employing traditional agarose gel electrophoresis and EtBr staining. Double-stranded RNA, purified from N. occidentalis 37B, was used to synthesize cDNA and for the development a cDNA library in Escherichia coli. Recombinant plasmids were analyzed by restriction enzyme digestion, and specific cDNA clones were selected by means of a 32P-labelled probe prepared from SMoV dsRNA. After a miniprep step, the plasmids were digested and the length of the inserts was determined to be between 200 and 2000 bp. The largest inserts were selected and used for further research. Hybridization experiments confirmed the specificity of the cloned material for SMoV dsRNA.

Improved Method to Retrieve DNA from Dried Silver-stained Polyacrylamide Gels

Differential display analysis (DDA) has become a useful and popular technique to identify differentially expressed genes in many tissues and cell types. Recovery of DNA fragments, however, from silver-stained nondenaturing polyacrylamide gel is difficult. Numerous techniques for extracting DNA from the polyacrylamide gel have been reported, including using untreated dried polyacrylamide flakes (1) , leeching with water ((2)) , excision and boiling in 1× PCR buffer (3) , and heating overnight in 0.5 mol/L ammonium acetate (4) . The potential problems with these techniques are that inhibitors can be carried over into the PCR reaction and that inconsistent preparation can lead to poor re-amplification. In addition, most of these techniques require excessive thermal cycling to re-amplify the fragment. We developed a technique in which the DNA is electroeluted out of the acrylamide onto a DEAE membrane. With this technique, multiple samples can be processed and purified simultaneously, leading to reproducible …

Development of a Highly Polymorphic STR Marker for Identity Testing Purposes at the Human Androgen Receptor Gene (HUMARA)

We developed a non-isotopic method which improves the technical quality of the X-linked HUMARA locus typing process. The use of formamide and a low concentration of acrylamide increased resolution and sharpness of HUMARA alleles in silver-stained polyacrylamide gels. In addition, the construction of an allelic ladder containing amplified sequence of 9 alleles (even-numbered alleles) of the HUMARA locus, allows confident, rapid and precise assignment of discretely defined alleles. Allele and genotype frequencies for the HUMARA locus were determined in a French Canadian population sample. Observed genotype frequencies in females conformed to Hardy-Weinberg expectations. Furthermore, the HUMARA locus is highly polymorphic with 18 observed alleles and an heterozygosity value of 89.3%. Also, this locus has average powers of discrimination of 97.8% and 88.7% for testing samples of female and male origin, respectively. In the French Canadian population, the average probability of excluding a random man as the father in paternity analysis when both mother and daughter are tested for this locus is 88.0%. Together, the results indicate that the HUMARA locus provides a highly discriminatory system that is appropriate for the purposes of forensic identification and paternity testing involving a female child.

A high sensitivity-nested PCR assay for BHV-1 detection in semen of naturally infected bulls

Several different PCR protocols for the detection of Bovine herpesvirus-1 (BHV-1) in bovine semen, are available in the literature. Most of them are rather laborious and the majority were performed on laboratory samples, artificially contaminated semen or semen provided from experimentally inoculated animals. Furthermore, to obtain higher levels of sensitivity, additional dot-blot procedures are frequently necessary. We describe the detection of BHV-1 in bovine semen and the supernatant of cell cultures with titres of 0.001 TCID 50/50 μl by a nested PCR assay, with no further hybridization procedures. The high sensitivity was achieved by filtering the semen samples on chromatography columns before DNA extraction, by using two pairs of primers in a nested PCR and by evaluating the amplification products on silver-stained polyacrylamide gels. Specificity of the amplified fragments was confirmed by RFLP and sequence analysis of the PCR products. This nested PCR procedure was performed in parallel with viral isolation (VI) on 101 semen samples provided from naturally infected bulls housed at an artificial insemination centre. The nested PCR was shown to be more sensitive, faster and easier to perform than the standard VI test. To our knowledge, it is the most sensitive PCR test for BHV-1 detection in bovine semen and could be easily used for routine diagnosis.

Characterization of chemotactic responses and flagella of Hyphomicrobium strain W1-1B.

Motile swarmer cells of Hyphomicrobium strain W1-1B displayed positive chemotactic responses toward methylamine, dimethylamine, and trimethylamine but did not display significant chemotactic responses towards methanol and arginine. Electron micrographs of negatively stained intact flagellar filaments indicated a novel striated surface pattern. The flagella were composed of two proteins of 39 and 41 kDa. Neither protein was a glycoprotein as determined by Schiff's staining and by enzyme immunoassay. Protein fingerprints visualized from silver-stained polyacrylamide gels and Western blots of protease-digested samples indicated that the two proteins were similar but not identical. Monoclonal antibodies prepared to the complex flagella of Rhizobium meliloti cross-reacted with the striated flagella of Hyphomicrobium strain W1-1B; however, these antibodies did not cross-react with smooth-surface flagella. These results suggest that complex and striated flagella possess homologous epitope regions.

Xanthomonas campestris pv. campestris lpsI and lpsJ genes encoding putative proteins with sequence similarity to the alpha- and beta-subunits of 3-oxoacid CoA-transferases are involved in LPS biosynthesis.

A 2.1-kb SmaI-EcoRI DNA fragment upstream of the xanA and xanB genes of Xanthomonas campestris pv. campestris carries two ORFs encoding putative proteins with sequence similarities to the alpha- and beta-subunits of 3-oxoacid-CoA transferases. The two ORFs were termed lpsI and lpsJ because strains carrying appropriate mutations showed an autoagglutination phenotype and because lipopolysaccharides of these mutant strains were altered according to silver-stained polyacrylamide gels. The monosaccharide composition of the exopolysaccharide xanthan produced by lpsI and lpsJ mutants remained unchanged.

Co-purification of a ribonuclease and human chorionic gonadotrophin β-core protein from human urine and displacement of 125I-human luteinizing hormone from Candida albicans binding sites by ribonucleases

An 18 kDa pregnancy urine protein preparation, purified to apparent electrophoretic homogeneity as judged by silver-staining of polyacrylamide gels, inhibited binding of 125I-hLH (human luteinizing hormone) to Candida albicans microsomes, reacted with monoclonal and polyclonal antibodies raised against human chorionic gonadotrophin (hCG) β-core protein and exhibited ribonuclease (RNase) activity. Eleven of the 12 amino acids at the N-terminus of a protein in this preparation were identical to those of the N-terminus of human non-secretory ribonuclease. These results indicate co-purification of hCG β-core with a RNase. An 18 kDa RNase was also purified from a commercial hCG preparation (Chorulon). However, no RNase activity was detected in a highly purified commercial preparation (Profasi). Three commercial RNase preparations displaced 125I-hLH from C. albicans binders at extremely low concentrations (<0.001 μg/ml RNase) whereas only slight displacement of 125I-hLH from sheep luteal binding sites was observed with very high concentrations of the RNases (100 μg/ml RNase). The co-purification of hCG β-core and RNase from pregnancy urine and the displacement of 125I-hLH from C. albicans binding sites by RNases may be related to the close relationship that has been identified between mammalian RNase inhibitors and the extracellular domain of gonadotrophin receptors. The presence of RNase in commercial preparations of gonadotrophins should be borne in mind during any investigations that involve impure preparations of these hormones.

Comparison of Chlamydia psittaci from cats with upper respiratory tract disease by polymerase chain reaction analysis of the ompA gene

Conjunctival swabs were taken from 168 cats with clinical signs of acute or chronic upper respiratory tract disease and tested for Chlamydia psittaci by the polymerase chain reaction (PCR) to...

A simple method for isolation of PCR fragments from silver-stained polyacrylamide gels by scratching with a fine needle

▼PCR-based DNA and RNA fingerprinting techniques, including arbitrarily primed PCR (AP-PCR) and differential display reverse transcriptase PCR (ddRT-PCR), represent comprehensive genetic screening methods (Ref. 1). Recently, sensitive nonradioactive detection techniques have been introduced for visualization of fingerprints using silver staining of the PCR products after polyacrylamide gel electrophoresis (Ref. 2, 3). Characterization of polymorphic PCR fragments depends on prior isolation of the corresponding bands from stained and dried gels. In most protocols, PCR products of interest have been cut out of the dried gel complete with the paper support, and DNA eluted by boiling in elution buffer (Ref. 4). Reamplification of the eluted PCR fragments has been successful in up to 75% of the experiments. However, additional bands neighbouring the fragment of interest were frequently coamplified. To avoid coamplification of contaminating bands, the whole procedure of cutting, boiling and reamplification was repeated up to five times by some protocols (Ref. 5). Here we demonstrate simple and rapid reamplification of PCR products after isolation by gently scratching over the silver-stained band using a sterile disposable needle [Microlance 3 (Becton-Dickinson; 0.7 mm)] prewetted with PCR mastermix, without destroying the gel. After scratching the band from the gel, the needle is placed into 20 μl standard PCR reaction mix for 1 min and then discarded. The scratched PCR products are reamplified by 30 PCR cycles (Fig. 1). Using this scratching technique, we are able to reamplify PCR products of interest in more than 90% of the experiments. In addition to the simplicity and speed of

Epidermal growth factor receptor gene and c- erbB-2 gene amplification in ovarian cyst fluid

To test for a significant difference between functional and neoplastic ovarian cyst with respect to epidermal growth factor (EGF) receptor and c-erbB-2 proto-oncogene amplification rates. We determined amplification of EGF-receptor and c-erbB-2 genes by differential polymerase chain reaction (PCR) on 138 ovarian-cyst aspirates. The semiquantitative differential PCR is based on simultaneous co-amplification of a target gene and a reference gene. Amplification rates were detected by densitometry of silver-stained polyacrylamide gels and were scored as single-, low-, or high-copy numbers. Wilcoxon ranked sum test, Pearson correlation coefficient, and multiple logistic regression were used to evaluate the differences in oncogene amplification and to predict histology. There were 71 (51.5%) women with functional cysts, whereas 67 (48.5%) had benign (n = 59) or malignant (n = 8) tumors. Low-copy (two- to fourfold copy numbers) EGF-receptor gene amplification was found in 22 of 67 (33%) women with neoplastic cysts, but in only eight (11%) of those with functional cysts. Neoplastic histology differed significantly from functional histology in correlation to EGF-receptor low-copy gene amplification (r = .279, P < .001). There was no significant difference in c-erbB-2 gene amplification with respect to functional and neoplastic histology (r = .083, P = .32). Low-copy EGF-receptor gene amplification seems to be a market for neoplastic histology. Epidermal growth factor receptor gene amplification may be involved in proliferation and growth in an early stage of tumorigenesis. However, further studies are required to investigate this gene structure abnormality on a gene function level. Amplification of c-erbB-2 proto-oncogene does not appear to be a common factor in the development of ovarian tumors.

A PCR-based test suitable for screening for fragile X syndrome among mentally retarded males

Ever since the identification of the genetic cause of fragile X syndrome as the expansion of an unstable trinucleotide sequence, several diagnostic strategies have evolved from molecular studies. However, we still lack a simple test suitable for population screening. We have therefore developed a nonisotopic polymerase chain reaction (PCR)-based technique for the identification of fragile X full mutations among men, with easy visualization of the PCR products on silver-stained polyacrylamide gels. The technique consists of PCR amplification with primers that flank the trinucleotide repeats, with a product of 557 bp for the (CGG)29 allele. Conditions were established such that full mutations failed to amplify and were thus identified with 98% sensitivity compared with Southern blot analysis. To produce an indispensable internal control we added to the reaction a third primer, internal to this fragment, allowing the multiplex amplification of a monomorphic band corresponding to a CG-rich stretch 147 bp upstream of the polymorphic region. In trials involving 41 patients and 74 controls, the PCR-based test here described showed specificity of more than 98.6%, accuracy of 99% and a sensitivity of 98%. Thus, although not suitable for medical diagnosis, it constitutes a useful tool for screening for the fragile X syndrome in populations of mentally retarded males.

Purification of lipopolysaccharide-binding protein from bovine serum

Lipopolysaccharide-binding protein (LBP) plays a central role in presentation of bacterial-derived lipopolysaccharide (LPS; endotoxin) to leukocytes such as macrophages and neutrophils. Interaction of LBP with LPS is significant because LBP-LPS complexes promote activation of leukocytes and the immune system, which results in enhanced secretion of a spectrum of proinflammatory cytokines. An improved, simplified method was used to purify bovine LBP from serum. Methodology consisted of ion-exchange chromatography using Bio-Rex 70 resin, followed by gel-filtration chromatography (Sephacryl S-200 resin) of a selected ion-exchange fraction (0.22-0.50 M NaCl). Densitometric scans on silver-stained polyacrylamide gels of chromatographically-derived proteins indicated up to 88.7% purity of the resultant 64 kD protein (bovine LBP) in the cleanest fractions. The isoelectric point of bovine LBP was determined to be 6.8. Identity of the protein was substantiated by western-blot analysis, and by N-terminus amino acid sequence analysis with favorable comparison to published sequence data from rabbit, human, and murine LBP Identity was corroborated by use of purified bovine LBP in bioassays which demonstrated enhanced tissue factor expression of LPS (1 ng ml −1)-stimulated bovine alveolar macrophages. Tissue factor expression was inhibitable in these assays using anti-CD14 monoclonal antibodies, which is also consistent with LBP-mediated activation of cells. When bovine LBP was heated at 56°C for 30 min, the biological activity was reduced by 50% in the macrophage-based bioassays. Biological activity of bovine LBP was completely destroyed by heating at 62°C for 30 min, which compared favorably with data resulting from use of fetal bovine serum.

Cloning and mapping of variety-specific rice genomic DNA sequences: amplified fragment length polymorphisms (AFLP) from silver-stained polyacrylamide gels.

An efficient technique for cloning DNA from silver-stained denaturing polyacrylamide gels was developed to allow the isolation of specific bands obtained from selective restriction fragment amplification (SRFA). This method proved as reliable as cloning radioactively labelled SRFA bands from the same gels. Rice DNA was used as a template, both with and without [32P]dCTP, using the same PCR profiles. Amplified products were separated using denaturing polyacryamide gel electrophoresis and visualized either by silver staining of gels or by autoradiography of 32P-labelled products. We cloned specific polymorphic SRFA bands directly from the denaturing polyacrylamide gels with one round of PCR amplification and confirmed that the sequences of the bands from silver-stained gels were identical to the corresponding 32P-labelled bands. The bands that were chosen represented amplified fragment length polymorphisms (AFLPs) between japonica and indica rice varieties. We studied the ability of two cloned AFLP bands to serve as heritable genetic markers by mapping them as RFLPs in an interspecific rice population and found that they represented single-copy DNA at unique loci in the rice genome.

Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels.

Proteins from silver-stained gels can be digested enzymatically and the resulting peptide analyzed and sequenced by mass spectrometry. Standard proteins yield the same peptide maps when extracted from Coomassie- and silver-stained gels, as judged by electrospray and MALDI mass spectrometry. The low nanogram range can be reached by the protocols described here, and the method is robust. A silver-stained one-dimensional gel of a fraction from yeast proteins was analyzed by nano-electrospray tandem mass spectrometry. In the sequencing, more than 1000 amino acids were covered, resulting in no evidence of chemical modifications due to the silver staining procedure. Silver staining allows a substantial shortening of sample preparation time and may, therefore, be preferable over Coomassie staining. This work removes a major obstacle to the low-level sequence analysis of proteins separated on polyacrylamide gels.

Quantitation of the water channel protein aquaporin (CHIP28) from red blood cell membranes by densitometry of silver stained polyacrylamide gels.

A protein determination procedure which involves the densitometry of silver stained polyacrylamide gels is described. It involves calibration with bovine serum albumin and molecular weight markers on the same gel with the protein to be quantitated. The procedure is simple, rapid, reproducible and accurate and is more sensitive than other procedures for protein determination. The procedure is particularly useful in quantitating proteins purified in small amounts since the determination can be performed on the same gel used to check the purification. It avoids interference by detergents and other substances usually present in solutions of purified proteins. The procedure has been applied to the quantitation of a recently identified protein, aquaporin (CHIP28), assumed to be a major water channel in the red blood cell membrane. A quantitative analysis of a purified fraction of this protein shows that the 28 kDa component represents approximately two thirds of the protein content of the sample, with the remainder comprising a glycosylated, high molecular mass component. The procedure may be useful for quantitating proteins revealed on silver stained gels and could be included as a standard part of any protocol for protein purification.

Rapid Removal of Nonspecific Background in Silver-Stained Polyacrylamide Gel

Rapid Removal of Nonspecific Background in Silver-Stained Polyacrylamide Gel

Use of nondenaturing silver-stained polyacrylamide gel analysis of polymerase chain reaction amplification products for the differential diagnosis of Leptospira interrogans infection

A 285-bp DNA fragment was amplified using the polymerase chain reaction from 38 Leptospira serovars of six different genomic species. The fragments amplified exhibited differential mobilities on nondenaturing polyacrylamide gels resulting from sequence-dependent conformational alterations. Leptospira interrogans serovars could be distinguished from those of other species on this basis.

Double-differential PCR for gene dosage estimation of erbB oncogenes in benign and cancer tissues and comparison to cellular DNA content

Competitive and differential quantitative PCR methods circumvent the limiting factors of PCR which cause poor reproducibility. We describe the development and performance evaluation of another quantitative PCR method, double-differential PCR (ddPCR). The ddPCR method comprises the co-amplification of the single-copy gene HBB, the erbB-1, erbB-2 and erbB-3 oncogenes and the second single-copy reference gene SOD2 under equal reaction conditions. The ratio of band intensities of the PCR products in silver-stained polyacrylamide gels expresses the average gene copy number (AGCN) per cell of the erbB oncogenes. The coefficient of variability (CV) was less than 25% for an AGCN of 1. The PCR data were in correlation to the results from dot blotting. DNA image analysis did not reveal any correlation between DNA content and gene dosage deviation of the erbB oncogenes. The method was applied to normal breast tissue, benign breast diseases, breast cancer tissue and lymph node metastases. We suggest this method as being reproducible, low cost and rapid, and therefore suitable for clinical studies on erbβ oncogene dosage estimation.

HLA-DQA1 and Amelogenin Coamplification: A Handy Tool for Identification

A protocol for HLA-DQA1 and gender identification by single amplification is described. The use of the commercial HLA-DQA1 amplification kit (Perkin Elmer) permits a positive response for sex determination by adding primers for a short sequence on the first intron of Amelogenin gene. The suggested amplification protocol results in PCR products easily and clearly detectable on ethidium bromide stained agarose gel or silver stained polyacrylamide gel. In both gels the HLA-DQA1 observations at 242-239 bp are accomplished with a single band at 106 bp in females and a doublet 112-106 bp in males. HLA-DQA1 reverse dot-blot hybridization is unaffected by the presence of X and Y amplified fragments.

Rapid purification of cotton seed membrane-bound N-acylphosphatidylethanolamine synthase by immobilized artificial membrane chromatography

N-Acylphosphatidylethanolamine synthase (NAPES) is a membrane-bound enzyme present in cotton seedlings at a concentration of ⩽0.02% of the total protein. NAPES was purified to electrophoretic homogeneity in a single chromatographic step using immobilized artificial membrane (IAM) chromatography. The IAM column used for NAPES purification was etherIAM.PE C10/C3 and this surface contains a monolayer of immobilized phosphatidylethanolamine (PE). Since PE is an analogue of the natural substrate for NAPES, etherIAM.PE C10/C3 columns function as an affinity column for this enzyme. Detergent-solubilized microsomal proteins from cotton were loaded on to the etherIAM.PE C10/C3 column and eluted with buffered mobile phases containing 0.2 m M dimyristoylphosphatidylethanolamine (DMPE) and 2 m M dodecylmaltoside. Little NAPES functional activity eluted if DMPE was removed from the mobile phase. Mobile phase DMPE is also a substrate for NAPES, and therefore both the mobile phase and IAM surface contains NAPES substrates. Mobile phase DMPE may function as both a surfactant-type affinity displacing ligand effecting protein elution and also a stabilizing factor of NAPES functional activity. The loading capacity on semi-preparative etherIAM.PE C10/C3 (6.5 × 1.0 cm) columns was ca. 5 mg of total detergent solubilized microsomal proteins, and protein recovery was quantitative. This one-step IAM purification of NAPES resulted in a single band on silver-stained polyacrylamide gels, and 3940 fold increase in NAPES specific activity. The molecular mass of the purified NAPES protein is 64 000. 125I labeled [12-(4-azidosalicyl)amino]dodecanoic acid is a photoreactive fatty acid substrate of NAPES that was used to confirm protein purity.