Fast And Sensitive Silver Staining Of Dna In Polyacrylamide Gels Pdf
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In pathology , silver staining is the use of silver to selectively alter the appearance of a target in microscopy of histological sections ; in temperature gradient gel electrophoresis ; and in polyacrylamide gels.
- Fast and sensitive silver staining of DNA in polyacrylamide gels
- Silver staining
- Silver staining of DNA in polyacrylamide gels
- Silver Staining Protocol
Fast and sensitive silver staining of DNA in polyacrylamide gels
Nucleic acids can be detected at the picogram level using a quick and simple silver staining method 2. Using very thin polyesterbacked polyacrylamide gels, a further simplified protocol was compared to other widely used silver staining procedures. The improved protocol described here was the most sensitive, the fastest to perform, and had relatively few steps and reagents. This method also produced the least number of staining artifacts and offered images of high contrast. This is a preview of subscription content, access via your institution. Rent this article via DeepDyve.
Silver staining is a powerful technique for protein identification in gels as silver binds to chemical sidechains of the amino acids, including the carboxyl and sulfhydryl groups. It was introduced in and later adapted for protein separation from the polyacrylamide gel electrophoresis. The nucleation sites in proteins promote the reduction of silver ions by formaldehyde into microscopic grains of elemental silver, enabling their detection. The treatment does not modify the tertiary structure of the proteins. It has widespread applications owing to its high sensitivity, which typically is two orders of magnitude greater than the Coomassie and Ponceau staining methods and could even detect as little as 2 ng protein.
Protein extraction from Polyacrylamide Gel. Proteomics Sample Preparation Protein Precipitation. Common contaminants, including salts, solvents, 2-mercaptoethanol, amino acids and DNA, are well tolerated in this assay. The assay can detect from 4 to 80 pmol of biotin in a sample. The assay can be performed in the presence of detergents, urea and reducing agents. Effective range for the assay is 0.
Silver stains permit the detection of nanogram amounts of proteins and nucleic acids in gels or membranes. These silver stains have been adapted from histological and photographic photochemical protocols. The basic mechanism of the visualization of protein and nucleic acids by silver staining involves the reduction of ionic to metallic silver. Staining properties of individual amino acids, homopolymers, and small peptides, have been used to demonstrate the importance of the basic amino acids, lysine and histidine, and the sulfur containing amino acids in the silver staining of proteins while the purines have proven to be important in the staining of nucleic acids. Many silver stains demonstrate reproducible curvilinear relationships between silver densities and protein and nucleic acid concentrations. Their sensitivity and reproducibility permits their use in quantitative analysis.
Silver staining of DNA in polyacrylamide gels
The photochemically derived silver stain of nucleic acids in polyacrylamide gels originally described by Merril et al. Detection limits for double-stranded DNA fragments from HaeIII endonuclease digests of phage phi X 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.
To explore why some oligonucleotides in denaturing polyacrylamide gel could not be silver-stained, different oligonucleotides were analyzed using denaturing polyacrylamide gel electrophoresis stained with silver and asymmetric cyanine. It was unexpected that oligo dT was hard to be silver-stained. Moreover, the silver staining of an oligonucleotide containing base T could be partially or completely inhibited by base T. The inhibition of silver staining by base T was a competitive inhibition which could be affected by the amounts of the argyrophil nucleobase and base T, the cis-distance between the argyrophil nucleobase and base T, and the gel concentration.
Here, we report a simple and low-cost silver staining protocol which requires only three reagents and 7 min of processing, and is suitable for fast generation of high-quality SSR data in the genetic analysis. Simple Sequence Repeat SSR is one of the most effective markers used in plant and animal genetic research and molecular breeding programs. Silver staining is a widely used method for the detection of SSR markers in a polyacrylamide gel.
Silver Staining Protocol
A comparison of silver staining protocols for detecting DNA in polyester-backed polyacrylamide gel. Eight silver-staining protocols were applied to detect DNA in polyester-backed gels to select the optimal. Results showed important differences in staining quality and that four methods were well-suited for TGGE gels due to high sensitivity and low background, including the Bassam et al. In the past decade, temperature-gradient gel electrophoresis TGGE technique has been employed as a powerful tool for investigating microbial diversity in various environments 7, 10, 11, 13, Due to high sensitivity and low toxicity with the use of very simple and cheap equipments and chemicals 4 , the silver staining methods are widely used for DNA visualization in polyacrylamide gels and fall into two categories based on the reagent used for silver impregnation. Alkaline methods use diamine or ammoniacal silver solutions for gel impregnation and dilute acid solutions of formaldehyde for image development. In contrast, acidic methods impregnate with silver nitrate in a weakly acidic milieu and use formaldehyde to reduce silver under alkaline conditions 3.
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