A novel [Ag(NH3)2]+ probe for chemiluminescent imaging detection of proteins after polyacrylamide gel electrophoresis

The development of a novel [Ag(NH3)2]+ probe chemiluminescence (CL)-based imaging method for the detection of various proteins after PAGE is described. The detection is based upon the probe [Ag(NH3)2]+ catalyzing the CL reaction of the luminol-potassium persulfate system. The proposed method detects various proteins labeled by [Ag(NH3)2]+ and expands the application scope to SDS gels. It also detects proteins directly in polyacrylamide gels, without tedious transferring procedures. Furthermore, successful identification of proteins by peptide mass profiling using ionization MS was easily performed, and no pretreatments of gel prior to digestion are needed. Detection limits for standard marker proteins match CBB-R250 staining and the linear dynamic range is superior to CBB-R250 staining and silver staining. The CL imaging conditions, including luminescent reagents, silver ion concentration, the ammonia-controlled system and the washing reagents parameters have also been optimized.     

Yet another improved silver staining method for the detection of proteins in polyacrylamide gels

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.     

Development of polyacrylamide gels that improve the separation of proteins and their detection by silver staining

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.     

A method for quantifying radioactivity associated with protein in silver-stained polyacrylamide gels.

A method is described in which individual proteins labeled with weak beta-emitting radionuclides, separated by polyacrylamide gel electrophoresis, and stained with silver are released from the gel by the use of the periodate soluble cross-linking agent N,N'-dialyltartardiamide. The radioactivity can then be quantitated using liquid scintillation counting. The method is shown to be insensitive to reasonable variations in the intensity of staining as well as the gel volume over a practical range of gel slices. Recovery from the gel is extremely good with 93% of the counts associated with 14C-labeled proteins of known radioactive concentration being recovered. Analysis of a complex mixture of 3H-labeled proteins indicates resolution similar to that obtainable by autoradiography without the problems associated with quenching of autoradiographic signal by the staining procedure. The method is used to determine the amount of fucose and mannose incorporated into a putative cell adhesion protein during development of the cellular slime mold Dictyostelium purpureum.     

Silver and Cyanine Staining of Oligonucleotides in Polyacrylamide Gel

To explore why some oligonucleotides in denaturing polyacrylamide gel could not be silver-stained, 134 different oligonucleotides were analyzed using denaturing polyacrylamide gel electrophoresis stained with silver and asymmetric cyanine. As a result, we found that the sensitivity of oligos (dA), (dC), (dG) and (dT) to silver staining could be ranged as (dA) > (dG) > (dC) > (dT) from high to low. 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. The changes of the intensity of an oligonucleotide band caused by the changes of DNA base composition were diverse and interesting. The intensity of some oligonucleotide bands would significantly change when the changes of DNA base composition accumulated to a certain extent (usually ≥ 4 nt). The sensitivity of cyanine staining of ≤ 11-nt long oligonucleotides could be enhanced about 250-fold by fixing the gels with methanol fixing solution.     

Sensitive, quantitative, and fast modifications for Coomassie Blue staining of polyacrylamide gels

In spite of the high sensitivity of silver staining and the wide dynamic range of various fluorescent detection methods, Coomassie Brilliant Blue staining is still the most widely used protein detection technique for proteins separated by polyacrylamide gel electrophoresis. There are several reasons: Low price, Visible with the eye, Desk top scanners can be employed for image acquisition, Better for quantitative analysis than silver staining, Possible modifications for fast or highly sensitive staining, Mass spectrometry compatible.     

微量蛋白检测中不同银染方法的比较与分析

随着生物化学技术的不断发展,作为检测SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)中微量蛋白的银染方法也在不断改进和发展.采用4种不同的银染方法检测不同含量的牛血清白蛋白,结果显示单纯的银染过程中如果使用戊二醛固定会使蛋白检出更快速灵敏,而结合考马斯亮蓝的复合银染则较单纯银染灵敏度提高了5～7个数量级.     

About the mechanism of interference of silver staining with peptide mass spectrometry

The mechanism by which silver staining of proteins in polyacrylamide gels interferes with mass spectrometry of peptides produced by proteolysis has been investigated. It was demonstrated that this interference increases with time between silver staining and gel processing, although the silver image is constant. This suggested an important role of the formaldehyde used in silver staining development in this interference process. Consequently, a formaldehyde-free staining protocol has been devised, using carbohydrazide as the developing agent. This protocol showed much increased peptide coverage and retained the sensitivity of silver staining. These results were however obtained at the expense of an increased background in the stained gels and of a reduced staining homogeneity.     

A mechanically strong matrix for protein electrophoresis with enhanced silver staining properties.

Duracryl is a mechanically strong and elastic acrylamide-based matrix, useful for a wide variety of electrophoretic applications. The matrix is stable as a refrigerated solution for one year. Upon addition of appropriate catalysts, Duracryl forms a polymer-reinforced polyacrylamide gel matrix suitable for electrophoresis. The polymer-reinforced gel is superior to conventional polyacrylamide gels in terms of mechanical strength, elasticity and protein silver staining properties. Protein detection sensitivity by silver staining, as well as the linear response of silver deposition versus protein load, is equivalent to standard acrylamide/N,N'-methylene bisacrylamide gels. Additionally, the silver staining properties of the Duracryl matrix result in proteins appearing as monochromatic shades of grey instead of red, brown and yellow, as is the case of conventional polyacrylamide matrices. Monochromatic shades of grey are more suitable for image analysis and densitometry. The matrix is compatible with standard electroblotting and protein N-terminal sequencing procedures. Low acrylic acid content and conductivity allow incorporation of the matrix into isoelectric focusing gels. The matrix was found not to alter polypeptide migration relative to the standard acrylamide/N,N'-methylene bisacrylamide matrix.     

Detection of fluorescence dye-labeled proteins in 2-D gels using an Arthur 1442 Multiwavelength Fluoroimager

Labeling of proteins with SYPRO Orange, SYPRO Red, and SYPRO Ruby after 2-D polyacrylamide gel electrophoresis (PAGE) using plastic-backed immobilized pH gradient (IPG) strips and precast SDS polyacrylamide gels was tested. Protein spots were detected using an Arthur 1442 Multiwavelength Fluoroimager. The labeling methods described allow detection of proteins both after isoelectric focusing (IEF) and PAGE with a sensitivity higher than or comparable to standard silver staining methods. In addition to the post-labeling methods mentioned above, pre-labeling with the cysteine-specific fluorophore monobromobimane before 2-D PAGE is a sensitive, fast, and cost-effective alternative to existing staining protocols.     

Influence of carbon dioxide on the impregnation of the nervous tissue by silver

It has been shown that 4% carbon dioxide (CO₂) in the air above reaction mixture inhibits the initiation of the formation of silver nanoparticles from complexes with biogenic amines (noradrenaline and serotonin). At the same concentration of CO₂ in the air above solution of AgNO₃, which is used for staining nerve tissues by the method of Golgi, neurons are preferentially stained, whereas at a concentration of 0.06%, vessels are stained. It is suggested that the entry of free silver ions to neurons is due to the inhibition of sites of initiation of silver nanoparticles in vessels at high CO₂ concentrations, while the lack of inhibition leads to silver precipitation in vessels at low CO₂ concentrations. It can be assumed that, for stable silver impregnation, the concentration of CO₂ must be controlled.     

Proteins C23 and B23 are the major nucleolar silver staining proteins.

To examine the silver staining proteins of Novikoff hepatoma nucleoli, the nucleolar proteins were separated on two-dimensional polyacrylamide gels with an isoelectric focusing first dimension and an acid-urea gel second dimension. The nucleoli were sequentially extracted with (1) 0.6 M potassium acetate, pH 5.5 and (2) 2 M potassium acetate — 5 M urea — 10 mM Tris, pH 7.5. The silver staining method used for the detection of silver binding proteins in gels was similar to that used to stain the nucleolar granules on microscope slides. Two major silver staining proteins were found which were identified as (molecular weight × 10^?3/pI) proteins C23 (100/5.3) and B23 (37/5.1). These two proteins are the major acidic proteins in Novikoff hepatoma nucleoli.     

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.     

Membrane proteins PmpG and PmpH are major constituents of Chlamydia trachomatis L2 outer membrane complex

The outer membrane complex of Chlamydia is involved in the initial adherence and ingestion of Chlamydia by the host cell. In order to identify novel proteins in the outer membrane of Chlamydia trachomatis L2, proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. By silver staining of the protein profile, a major protein doublet of 100-110 kDa was detected. In-gel tryptic digestion and matrix-assisted laser desorption/ionization mass spectrometry identified these proteins as the putative outer membrane proteins PmpG and PmpH.     

Analysis of genetic mutations in human lactate dehydrogenase-A(M) deficiency using DNA conformation polymorphism in combination with polyacrylamide gradient gel and silver staining

Human lactate dehydrogenase (LDH)-A mutant gene was analyzed by polymerase chain reaction - DNA conformation polymorphism (DCP). We used polyacrylamide gradient gel and silver staining procedures for DCP analysis and observed abnormal migration patterns in individuals heterozygous for LDH-A deficiency. Further sequence determination of the mutant alleles consistently resulted in detection of base substitutions, a G to T transversion at codon 328 (GAG----TAG), and synonymous substitutions at codon 115, 160 and 172. Such mutations were easily detectable using the DCP technique. The DCP technique using the polyacrylamide gradient gel and silver staining method seems likely to be useful for the rapid screening of mutations and for further genotype detection.     

The Physical Chemistry of Silver Staining

It has been shown that silver deposition plays a part in the silver staining process. From this it has been concluded that the rate of reduction of silver within and on histological structures is an important factor.

Some factors controlling the rate of reduction, such as the adsorption of silver hydroxide and ammonia, the affinity of silver for proteins, and the protective power of the gel structures have been pointed out.

Some simple applications of the ideas to silver staining have been given and two technics described, one making use of piperidine instead of ammonia, the other carrying out the reduction in the presence of the silver solution to facilitate deposition.     

The ultrasensitive silver "protein" stain also detects nanograms of nucleic acids

The ultrasensitive silver staining procedure developed for proteins also stains nanogram quantities of RNA and DNA in polyacrylamide gels. A gradient polyacrylamide gel system is described which separates proteins from 10⁴ to 10⁶ M_r, RNA from 5S to 23S and DNA from 0.4 to 21 Kb. The sensitivity of nucleic acid silver staining in this gel system considerably exceeds that of commonly used DNA and RNA dye-binding stains.     

Detection of a few picograms of DNA on polyacrylamide gels by silver staining

DNA fragments separated on polyacrylamide gels are silver stained in ethanolamine solution. The staining procedure can be completed in 3 1/2 h. Illumination of the gels on a black background increases the sensitivity of detection compared with the usual transillumination. The limit level of detection is 3-5 pg per band with a cross-sectional area of 5 mm2. Five to fifty picograms of DNA may be detected quantitatively by scanning the gels. The method will detect 0.1 to 1 ng per band of low-molecular-weight RNA components.     

Analysis of differences between coomassie blue stain and silver stain procedures in polyacrylamide gels: conditions for the detection of calmodulin and troponin C

It is reported that the conditions used in some silver stain procedures can fail to detect calmodulin, troponin C, and other proteins with similar physical properties. Conditions are described that allow the reproducible detection of these proteins. Two phenomena are described: (1) lack of protein staining when treatment with glutaraldehyde is omitted from the protocol, and (2) loss of small proteins from the gel matrix during prolonged washing procedures. These data directly demonstrate that the use of some silver staining protocols can result in misleading data in biological studies and provide an explanation for at least one class of proteins of how silver staining and Coomassie blue staining of gels can give different results.     

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.