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1.
Detection of single sequence repeat polymorphisms in denaturing polyacrylamide sequencing gels by silver staining 总被引:19,自引:0,他引:19
Large-scale use of molecular markers in plant breeding is limited by the throughput capacity for genotyping. DNA polymorphisms
can be detected in denaturing polyacrylamide gels indirectly by nucleotide labeling or directly by staining. Fluorescent-labeling
or radiolabeling requires sophisticated infrastructure not always available in developing countries. We present an improved
low-cost method for silver staining and compare it to 2 other methods for their ability to detect simple sequence repeat polymorphisms
in denaturing polyacrylamide gels bound to glass plates. The 3 procedures differed in their requirement for an oxidation pretreatment,
preexposure with formaldehyde during silver nitrate impregnation, inclusion of silver thiosulfate, and by their replacement
of sodium carbonate for sodium hydroxide to establish alkaline conditions for silver ion reduction. All methods detected the
same banding pattern and alleles. However, important differences in sensitivity, contrast, and background were observed. Two
methods gave superior sensitivity, detecting down to 1 μL of loaded amplification products. Our improved method gave lower
backgrounds and allowed reutilization of staining solutions. The use of thin (<1 mm) denaturing sequencing gels allows genotyping
of 60–96 samples within 4 h. Use of smaller loading sample volumes and reutilization of staining solutions further reduced
costs. 相似文献
2.
Silver staining DNA in polyacrylamide gels 总被引:2,自引:0,他引:2
This protocol describes a simple silver staining method used to visualize DNA fragments and other organic molecules with unsurpassed detail following traditional polyacrylamide gel electrophoresis (PAGE). Sensitivity rivals radioisotopic methods and DNA in the picogram range can be reliably detected. The described protocol is fast (approximately 1 h) and is implemented using readily available chemicals and materials. To achieve the sensitivity and visual clarity expected, quality reagents and clean handling are important. The updated protocol described here is based on the widely used method of Bassam et al. (1991), but provides improved image contrast and less risk of staining artefacts. 相似文献
3.
A selective method for staining urease activity bands in nondenaturing polyacrylamide gels is described. It is based on the deposition of silver at the urease bands after incubation of gels in the presence of urea and photographic developers. Its highly sensitivity (up to 0.015 enzyme units, corresponding to 5 ng of purified urease) is based on both the silver deposition enhancement methodology and the developers used. The selectivity of the procedure is based on the local pH increase catalytically produced by the enzyme in the presence of urea. The densitometric scan of the enzyme bands gives a linear response at least in the range 0.015-0.300 urease units. This selective staining method is about 2.5 times more sensitive than the standard silver staining of proteins, in terms of detectable urease amount. 相似文献
4.
Proteins in which some or all of the tyrosine side chains are post-translationally modified to dihydroxyphenylalanine have been found in several invertebrate phyla. In this paper we describe the unusual silver-staining properties of these 3,4-dihydroxyphenylalanine (Dopa)-proteins in silver-stained polyacrylamide gels. Our evidence suggests that the rapid silver staining of these proteins is due to the 3,4-dihydroxyphenol ring which is a highly effective reducing agent in the alkaline development conditions used in the final step of most silver-staining procedures. Normal proteins comprising the standard 20 amino acids and tyrosine on its own, do not reduce silver under these conditions. Pretreatment of the gels with acid-dichromate solutions abrogates the rapid staining of the Dopa-proteins. This rapid silver-staining technique will facilitate the rapid screening of many additional organisms for Dopa-proteins using sodium dodecyl sulfate gels and small amounts of tissue. 相似文献
5.
The very sensitive and reliable silver staining method to visualize proteins in polyacrylamide gels described by Wray et al. (Anal. Biochem. (1981) 118, 197-203) fails when the protein sample contains nucleic acids and/or metals. By washing the polyacrylamide gels in acetic acid and repeatedly in methanol immediately following electrophoresis and then using the procedure of Wray et al., many gels otherwise unstainable may be stained with a high degree of reliability. This method allows visualization of a minute amount of proteins in samples containing high amounts of DNA and metals. 相似文献
6.
A highly sensitive technique for staining DNA and RNA in polyacrylamide gels using silver 总被引:5,自引:0,他引:5
A technique is described for staining DNA in polyacrylamide gels with silver. It is rapid, requiring about 30 min for whole staining and development procedure, very simple and at least 20 times more sensitive than ethidium bromide for the staining of double-stranded DNA in polyacrylamide gels. This technique can also be applied for the staining of denatured, single-stranded DNA as well as RNA after their electrophoretic separation on polyacrylamide gels, having the same sensitivity as for double-stranded DNA fragments. 相似文献
7.
ABSTRACTMethyl green (MG) is an inexpensive, nonproprietary, traditional histological stain for cell nuclei. When bound to DNA and upon excitation with orange-red light, it fluoresces brightly in the far red region. We compared MG with ethidium bromide (EtBr), the conventional stain for DNA in gels, and Serva DNA stain G? (SDsG), a proprietary stain marketed as a safer alternative to EtBr for staining of electrophoresed DNA bands in agarose and polyacrylamide gels. DNA-MG fluorescence was recorded and 2.4 μg/ml MG produced crisp images of electrophoresed DNA after incubation for 10 min. Stain solutions were stable and detection limits for faint bands as well as relative densitometric quantitation were equivalent to EtBr. MG, EtBr and SDsG cost 0.0192, 0.024 and 157.5 US cents/test, respectively. MG is an effective stain for visualizing DNA in agarose and polyacrylamide gels. Its major advantages including low cost, comparable quality of staining, storage at room temperature, photo-resistance and low mutagenic profile outweigh its disadvantages such as staining of tracking dye and requirement for a gel documentation system with a red filter. 相似文献
8.
When cast in a polyacrylamide gel, whole lipopolysaccharide (LPS) and the lipid A fraction of LPS fromSalmonella typhimurium andEscherichia coli O111B4 reacted with the silver stain described by Tsai and Frasch [11]. However, the polysaccharide fractions released from the LPS by acid hydrolysis were not stained. This is inconsistent with the previously believed notion that the polysaccharide component is that which reacts with the silver stain. 相似文献
9.
Experimentally improved reliability of ultrasensitive silver staining of protein in polyacrylamide gels 总被引:16,自引:0,他引:16
Variations of the ultrasensitive silver staining method of B. R. Oakley, D. R. Kirsch, and N. R. Morris (Anal. Biochem.105, 361–363 (1980)) have been tested. It was established that the reliability of the method was greatly improved if (i) free silver was carefully washed out before reduction with formaldehyde; (ii) the extent of development was controlled by using methylamine to inactivate the formaldehyde; and (iii) the optimum quantity of ammonia, which was found to be 4 mol/mol of silver was used (this quantity was defined as that which titrates a particular amount of 1 n HCl). The time of preparation of the formaldehyde reducer was found not to be highly critical. In our hands the method can detect down to 0.1 ng of protein/5-mm slot. 相似文献
10.
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. 相似文献
11.
Detection of subnanogram amounts of RNA in polyacrylamide gels in the presence and absence of protein by staining with silver 总被引:3,自引:0,他引:3
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. 相似文献
12.
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). 相似文献
13.
Requirement for cysteine in the color silver staining of proteins in polyacrylamide gels 总被引:1,自引:0,他引:1
To determine whether cysteine residues have a contribution to the mechanism of color silver staining, we silver stained sodium dodecylsulfate polyacrylamide gel electrophoresis separations of proteins which have few or no cysteines. Proteins without cysteine stained negatively (yellow against a yellow background) with silver. Proteins with one or more cysteines stained orange, red, brown, or green/gray depending on the mole percentage of cysteine and whether they contained covalently attached lipids. The colors could not be correlated with the mole percentages of cysteine of these proteins indicating that some components other than cysteine affect the staining color of cysteine-containing proteins. Silver staining of amino acids, sugars, nucleotide bases, or lipopolysaccharide dot-blotted onto nitrocellulose paper implicated adenine, lipids, the basic amino acids, and glutamine, but not sugars or other amino acids in silver/protein complexes. 相似文献
14.
15.
Wei-Tao Cong Zhong-Xin Zhu Hong-Zhang He Cheng-Xi Jiang Li-Tai Jin Xiao-Kun Li 《Analytical biochemistry》2010,402(1):99-101
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. 相似文献
16.
Development of polyacrylamide gels that improve the separation of proteins and their detection by silver staining 总被引:18,自引:0,他引:18
Background staining that is associated with silver detection of proteins and nucleic acids in polyacrylamide gels has been shown to be due mostly to the amide groups in methylenebisacrylamide, a commonly used gel crosslinker. In attempts to reduce this background staining, eight existing crosslinking agents were tested. All of these proved to be unsuitable. Six new crosslinking agents were synthesized and tested. Of these, diacrylylpiperazine provided increased physical strength, improved electrophoretic separation of proteins, and silver staining detection of proteins with reduced background stain. 相似文献
17.
T Marshall 《Analytical biochemistry》1984,136(2):340-346
A much improved silver staining procedure for the detection of protein in polyacrylamide gels of 0.8-3.0 mm thickness is described. It achieves very high sensitivity (detecting less than 0.01 ng bovine serum albumin/mm2) by overstaining and subsequently removing nonspecific background stain using a modified, reliable destaining procedure. Maximum sensitivity follows prediamine equilibration in 0.1% (w/v) formaldehyde solution. With two-dimensional electrophoresis the improved staining procedure reveals greater than 200 polypeptides in unconcentrated human urine and greater than 150 polypeptides in a single human fingerprint. 相似文献
18.
Yet another improved silver staining method for the detection of proteins in polyacrylamide gels 总被引:11,自引:0,他引:11
Silver staining is very sensitive for detection of proteins in polyacrylamide gels and different procedures have been published. By combining and modifying some of the recipes, a very reproducible method, which is based upon staining with diamine complexes of silver, has been developed. The background staining is negligible and reduced silver does not precipitate on the gel surface. The technique works very well for sodium dodecyl sulfate-polyacrylamide gel electrophoresis in both homogeneous and in gradient gels as well as for two-dimensional (2-D) PAGE. It was possible to detect 1-10 ng of protein corresponding to approximately 50 pg/mm2, provided that a discontinuous buffer system was used, which gives sharp bands. 相似文献
19.
Detection of oxidases on polyacrylamide gels 总被引:2,自引:0,他引:2
Direct transfer of electrons, from the reduced flavin moiety of several oxidases to phenazine methosulfate and then to nitroblue tetrazolium, serves to detect activity of the oxidases of l-amino acids, urate, diamines, hydroxy acids (both long- and short-chain), aldehydes, and sarcosine. It is demonstrated that the method specifically detects oxidases and is more sensitive than other methods available. It is noted that, in the detection of certain dehydrogenases on polyacrylamide gels by the usual NAD-phenazine methosulfate-nitroblue tetazolium technique, one or more of the isozymes may actually represent the corresponding oxidase rather than the dehydrogenase in question. 相似文献