The most powerful interactions occur with carboxylic acid groups (Asp and Glu), imidazoles (His), sulfhydryls (Cys), and amines (Lys). Furthermore, the background staining was decreased, which led to the enhanced detection efficiency and sensitivity of low-concentration proteins. In this study, the TEM results revealed that the larger the silver ion particles, the more irregular the shape, allowing for particles deposition. It has been observed that the larger particles are more irregular in shape, as shown in Figure 7. Heukeshoven and Dernick also evaluated the silver staining of the basic homopolymers of arginine, histidine, and ornithine but not for polylysine [7]. The samples were then subjected to ultrasonication for 10 minutes [16]. Histidine was found to be the most significant amino acid for silver ion binding and efficient silver staining among all amino acids. First, 2ml of the solution was added at 25C temperature and the measurements were performed thricely. Next, 0.5% PVP (polyvinylpyrrolidone, molecular weight 58KD), 0.5% AMP (2-amino-2-methyl-propanol), and 0.5% Tween-80 were added, followed by adding 7.5% ethanol, 1.7% sodium acetate, 0.125% glutaraldehyde, and 0.2% sodium thiosulfate pentaerythritol and then soaked for 30 minutes. The black arrow is 0.5ng target protein. The advanced phenomenon can result in the enhanced detection efficiency and sensitivity of low-concentration proteins. (b) The original method with the AMP group. The morphology and structure of the silver particles in the silver dye reagent were observed and verified by TEM transmission electron microscopy technology. At present, the influence of the high-level structure of the protein on the silver staining effect cannot be determined. A protein concentration of 0.5ng in the black arrow also has been shown. Moreover, the unclear and high background makes it difficult to detect low-concentration proteins [8]. Although the exact cause of the final staining effect is complicated and dependent on several factors, this experiment reveals that using these reagents increases the size of the silver ion particles, as shown in Figure 8. Furthermore, 0.5% AMP, 0.5% PVP, 0.5% Tween-80 reagents significantly influenced the morphology, size, potential, and dispersion of silver ions. We thank the Shenzhen Science and Technology Innovation Commission for the Shenzhen Basic Research Technology Breakthrough (general project, grant number: JCYJ202103241113210027). It is also believed that the silver staining of protein bands is based on the combination of various groups in the protein (such as thiol and carbon group) with silver, resulting in the adhesion of silver ions to the surface of the protein, forming silver ion deposition, and encountering reducing agents that result in the precipitation of the color. The electrophoresis bands in this experiment are BSA protein (20ng, 50ng, and 100ng) and protein marker, from left to right. Large particles are often between 200 and 300nm in size but can reach 300nm, while small particles are typically less than 100nm in size. The authors declare that they have no conflicts of interest. The result of the same batch of detection has a lower background and clearer bands. The statistical software SAS 12.0 was used to calculate the average value for each group. The significant level dividing line was or 0.01 [13]. (a) Original treatment group. Numerous modifications to the silver staining procedure can alter the oxidation-reduction equilibrium, resulting in the visual representation of gel-separated proteins as positively or negatively stained bands [3]. (a) Original method without other reagents. Certain protein functional groups interact and bind with silver ions (from silver nitrate in the staining reagent). Aside from silver ions attached to proteins, silver ions in the vicinity of the protein are also required for the formation of bands. The black arrow is 0.5ng target protein which is difficult to distinguish in (b) and even cannot be seen in (a). Although the staining effect has been improved, the underlying explanation is still unknown. Silver-stained protein bands generally are dark brown or black with considerable variation in color intensity. Figure 3(a) shows the results obtained using the original untreated approach, while Figure 3(b) shows the results using the modified procedure with PVP, AMP, and Tween-80. The copper mesh upside was put down on the sample and suspended for 1.5 minutes. Several factors influence the efficiency and sensitivity of silver staining. Although the electric potential may not explain all the phenomena, it still reflects a certain trend for size of particles. (b) Xylene treatment group. This work presents a new approach to further enhance the protein silver staining detection technique. The reaction was then stopped by adding 2% duodenum edetate for 10 minutes, followed by adding double-distilled water for washing thricely (5 minutes each time). According to the obtained statistics results, PVP and AMP treatment increases the size of AgNO3 particles to varying degrees. PVP, AMP, Tween-80, and other reagents were used in this investigation to possibly influence the potential difference of silver ion particles, making it simpler for silver ion particles to form large particle deposits on the protein surface. Another element that influences the sensitivity of protein staining on gels is protein reactivity with silver ions. This study has been supported by funding. The reactivity of silver ions, and hence the sensitivity of protein detection, is also affected by protein structure. In previous studies, it has been revealed that a reducing agent or the concentration of silver ions modulated the size of the silver ion particles. (c) The original method with the Tween-80 groups. The particle potential analysis: (a) 0.5% AgNO3; (b) 0.5% AgNO3 and 0.5% Tween-80; (c) 0.5% AgNO3, 0.5% Tween-80, and 0.5% AMP; (d) 0.5% AgNO3, 0.5% Tween-80, and 0.5% PVP; (e) 0.5% AgNO3, 0.5% AMP, and 0.5% PVP; (f) 0.5% AgNO3 and 0.5% PVP; (g) 0.5% AgNO3 and 0.5% AMP; (h) 0.5% AgNO3, 0.5% AMP, 0.5% Tween-80, and 0.5% PVP. According to the glue observation, the proper amount of double distilled water was added to save the gel. The color variation has been attributed to diffractive scattering by silver grains of different sizes. In nearly all cases, adding these components to fixation, silver salt, or development solutions did not result in a well-defined enhancement of stained protein bands. Then, 10L of 2% phosphotungstic acid was dropped on the clean sealing film. Silver staining is an excellent technique for detecting proteins that are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The copper mesh and filter paper were clamped and sucked up the excess liquid. The quantitative findings of ultratrace BSA protein in an embodiment of the present invention are shown in Figure 4. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. In this study, it has been revealed that the staining ability of protein silver stain was increased by adding 0.5% of AMP, PVP, Tween-80, and xylene. Herein, we used commercially available BSA (bovine serum albumin) (Sigma fatty acid-free type) standard protein samples. The obtained results demonstrated that the smaller the potential, the larger the particle size. Target protein can be checked in both of B and C groups. At the termini of amino acid side chains, the amino and imidizole groups are capable of cooperating intramolecularly to bind silver ions, whereas the peptide bonding and N-terminal amino groups are not [3]. This is sufficient to demonstrate that PVP, AMP, and Tween-80 decrease the silver ion potential and increase the particle size, which makes it easier for the particles to aggregate on the protein surface as an alteration in ion potential significantly affects the process of silver staining [6]. The histidine ligands connected the silver ions by binding through the imidazole nitrogen atom on one hand, and the N atom of the NH2 group on the other [9]. (c) Xylene, PVP, AMP, and Tween-80 added group. Silurian materials were dyed with 2% phosphotungstic acid [17], and then, 10L of the sample was dropped on a clean parafilm. Metallic silver is deposited onto a gels surface at the positions of protein bands in this approach. Herein, we modified the recipe of silver staining, a very reproducible method, by adding AMP, PVP, Tween-80, and xylene to enhance the detection ability of protein staining. The presence of amino acid in protein structure affects the process of silver staining. This technique rapidly gained popularity owing to its high sensitivity as compared to other stains including the Coomassie Brilliant Blue R250 stains. Comparison of the effects of various additives. SDS-PAGE of serial dilutions of BSA protein. 2007, An et al. The procedure described above resulted in an increase in detection sensitivity. We compared various modifications to the protocol of silver staining and examined the vital reaction steps and the effects of various additives to fixation solutions, such as glutaraldehyde and formaldehyde, and to the solution of silver staining, such as oxidizing agents [3], copper salts [10, 11], ammonium nitrate [12], and NaOH. The staining process sequentially consists of protein fixation, sensitization, washing, silver impregnation, and finally development of the image. B. Bokhonov, L. P. Burleva, D. R. Whitcomb, and M. R. Sahyun, Electron microscope characterization of AgBr heterojunctions with silver carboxylates and their influence on the morphology of developed silver particles in thermally developed photomaterials,, P. Kumari, H. Rickard, and P. Majewski, Deposition of silver and gold nanoparticles on surface engineered silica particles and their potential applications,, G. D. Jay, D. J. Culp, and M. R. Jahnke, Silver staining of extensively glycosylated proteins on sodium dodecyl sulfate-polyacrylamide gels: enhancement by carbohydrate-binding dyes,, T. Rabilloud, L. Vuillard, C. Gilly, and J.-J. Among them, the composite treatment group 6 has the largest particle size and lower potential. Modification in Silver Staining Procedure for Enhanced Protein Staining, Department of Biomedical Engineering, Shenzhen Peoples Hospital (The Second Clinical Medical College, Jinan University, The First Affiliated Hospital, Southern University of Science and Technology), Shenzhen, 518020 Guangdong, China, P. S. Abdul-Rahman, B. K. Lim, and O. H. Hashim, Expression of high-abundance proteins in sera of patients with endometrial and cervical cancers: analysis using 2-DE with silver staining and lectin detection methods,, M. Chevallet, S. Luche, and T. Rabilloud, Silver staining of proteins in polyacrylamide gels,, C. R. Merril, Development and mechanisms of silver stains for electrophoresis,, H. Bartsch, C. Arndt, S. Koristka, M. Cartellieri, and M. Bachmann, Silver staining techniques of polyacrylamide gels Protein Electrophoresis,, G. Berson, Silver staining of proteins in polyacrylamide gels: increased sensitivity by a blue toning,, C. R. Merril and M. E. Pratt, A silver stain for the rapid quantitative detection of proteins or nucleic acids on membranes or thin layer plates,, B. L. Nielsen and L. R. Brown, The basis for colored silver-protein complex formation in stained polyacrylamide gels,, J. Heukeshoven and R. Dernick, Simplified method for silver staining of proteins in polyacrylamide gels and the mechanism of silver staining,, L. Mirolo, T. Schmidt, S. Eckhardt, M. Meuwly, and K. M. Fromm, pH-dependent coordination of agi ions by histidine: experiment, theory, and a model for sile,, R. C. Allen, Rapid isoelectric focusing and detection of nanogram amounts of proteins from body tissues and fluids,, C. R. Merril, D. Goldman, S. A. Sedman, and M. H. Ebert, Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins,, C. R. Merril, M. L. Dunau, and D. Goldman, A rapid sensitive silver stain for polypeptides in polyacrylamide gels,, M. R. de Campos, A. L. Botelho, and A. C. Dos Reis, Nanostructured silver vanadate decorated with silver particles and their applicability in dental materials: a scope review,, H. A. Goldberg and K. J. Warner, The staining of acidic proteins on polyacrylamide gels: enhanced sensitivity and stability of "stains-all" staining in combination with silver nitrate,, N. M. Pham, S. Rusch, Y. Temiz, H.-P. Beck, W. Karlen, and E. Delamarche, Immuno-gold silver staining assays on capillary-driven microfluidics for the detection of malaria antigens,, B. All reagents were made with premium-grade, which were mixed and boiled for 5 minutes before sample loading and electrophoresis. The morphology and structure of the silver particles in the silver dye reagent were observed and verified by TEM. The combination of PVP, AMP, and Tween-80 kits resulted in more stained protein bands. The functional groups of proteins must be exposed in order for silver ions to attach to them. (c) Treatment with PVP, AMP, and Tween-80 with xylene. The development process is nearly identical to that of the photographic film: silver ions are converted to metallic silver, yielding a brown-black color [18, 19]. Relevant technical principles and methods provide a reference for further improving the detection effect and ideas of silver staining technology. Hence, if large amounts of silver ions are deposited on the protein bands, it makes it easier to detect low-abundance proteins. Lawrence, Silver-staining of proteins in polyacrylamide gels: a general overview,, L. A. However, proteins that do not contain or rarely contain cysteine residues are sometimes negatively stained [4, 5]. The concentration and separation gels were 5% and 10%, respectively, with voltages of 80V and 140V. A 5X commercial SDS buffer (Tris-HCL PH6.8, 60mM; SDS 2%; bromophenol blue 0.1%; glycerol 25%; -mercaptoethanol 14.4mM) was used. However, when the technique was first established, it had various drawbacks, the most notable of which were a high background and frequent silver mirrors, as well as diminished sensitivity and reproducibility. A comparative evaluation of the effects of various additives has been carried out. Figure 3 shows the detection of BSA protein using electrophoretic staining. The detection sensitivity was significantly enhanced by 20200 times, allowing proteins as low as 0.1ng protein per band to be detected [1, 2]. Frequently, the additives resulted in an intensification of background staining. According to Nielsen and Browns observation, the basic amino acids lysine, arginine, and histidine, in both free orand homopolymeric forms, form colored complexes with silver reinforcing the role of the basic amino acids in silver staining [8]. The dye solution on the copper mesh was suspended for 2.5 minutes. We modified the traditional staining method by adding AMP, PVP, Tween-80, and xylene to enhance the detection ability of protein staining. The obtained results revealed that the use of 0.5% of AMP, PVP, Tween-80, and xylene improved the staining ability of protein silver staining, compared with the original method. Furthermore, the detection limit was estimated to be lower than 0.5ng per protein band. This study found that the morphological structure of silver particles is of great significance to the effect of protein silver staining technology and improving the sensitivity of silver staining. Silver staining is an excellent technique for detecting proteins that are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) due to its efficiency in detecting proteins present in nanograms. Detection of BSA protein using electrophoretic staining (a) Original treatment method without xylene. Figure 4(b) is whiter than the gel block on the right (Figure 4(c)), and the band with a protein concentration of 0.5ng in the black arrow on the right is the most obvious, and the color rendering effect is significantly improved. Except for the marker, the protein concentration of the band from left to right was 0.1ng, 0.2ng, 0.5ng, and 10ng. This result shows that treatment with PVP, AMP, and Tween-80 can increase the size of silver particles, facilitating the precipitation of silver ion particles on a large scale. Few studies, however, demonstrated that color variation is due to the formation of silver chromate deposits that are incorporated into formalin-fixed proteins. No related content is available yet for this article. PVP, AMP, Tween-80 also has an effect on silver ion shape, size, potential, and dispersion. Protein silver staining technology has higher sensitivity and is suitable for the detection of low-concentration proteins compared to other staining techniques including the Coomassie brilliant blue detection method. Furthermore, the size of silver particles deposited on the protein surface has a significant impact on the protein gels color development. A two-color 25-250KD protein marker was used as an electrophoresis label, followed by the gel documentation using the iBright Imaging System [1]. Herein, an appropriate amount of PVP, AMP, Tween-80, and xylene was added during the staining to advance the sensitivity of silver stain detection and reduce the staining background which may result in the improvement of the target fragment brightness and the silver staining effect. In this study, we used 0.5% of AMP, PVP, Tween-80, and xylene which led to the enhancement of the silver staining technique. Furthermore, the particle size and potentiometer were used to detect the particle size and potential difference of the silver ions in the prepared dyeing materials, and then, the morphology, transparency, and size of the dyed silver particles in different dyeing solutions were studied using a transmission electron microscopy (TEM). The visualization of protein bands appeared as spots at the site of reduction, and therefore, the image of protein distribution within the gel is based on the difference in oxidation-reduction potential between the free adjacent sites and gel area occupied by the proteins. Read the winning articles. Hence, the present study conducted a new modified method for silver-stained polyacrylamide gel in protein staining, which can solve the problems of high background and insufficient color development in the existing silver staining. Following electrophoresis, a full piece of gel was obtained; however, during staining, the gel was separated and stained separately. The present study was conducted to enhance the detection ability of the protein staining method. The gel was fixed with fixative solutions, i.e., ethanol (50%), glacial acetic acid (10%), and double-distilled water (40%) for 30 minutes, followed by gel treatment with ethanol (30%), sodium acetate (6.8%), and glutaraldehyde (0.6%) for 40min. Furthermore, the images also demonstrated the black spot which may be associated with the larger particle size of silver [3]. Butcher and J. K. Tomkins, A comparison of silver staining methods for detecting proteins in ultrathin polyacrylamide gels on support film after isoelectric focusing,. The obtained results suggested that our recipe for protein silver staining makes the glue block more transparent and improves the overall yellow condition of the glue block. The particle size and potential difference were measured using a Malvern particle size potentiometer (ZEN 3700). As such, traditional staining methods have a poor effect, and the background remains high, resulting in limited detection capabilities for low-abundance proteins. Other reagents can increase particle size to varying degrees. These results suggested a new idea for further improving the detection ability of protein silver staining. The average value was then used as the criterion after 10 minutes of stable measurements [1315]. Compared with the original method, the sensitivity was improved after adding additives. Moreover, Figure 3(c) shows the results obtained from the new method, employed with PVP, AMP, and Tween-80, followed by treatment with xylene. It can be performed with simple and inexpensive laboratory reagents, and the readout does not necessitate complicated and expensive equipment. 2009). PVP, AMP, and Tween-80 were then added. First, eight samples (A-H) of silver ion materials were prepared including (A) 0.5% AgNO3, (B) 0.5% AgNO3,0.5% Tween-80, (C) 0.5% AgNO3, 0.5% Tween-80, 0.5% AMP, (D) 0.5% AgNO3, 0.5% Tween-80,0.5% PVP, (E) 0.5% AgNO3, 0.5% AMP, 0.5% PVP, (F) 0.5% AgNO3, 0.5% PVP, (G) 0.5% AgNO3, 0.5% AMP, (H) and 0.5% AgNO3, 0.5% AMP, 0.5% Tween-80, 0.5% PVP. Article of the Year Award: Outstanding research contributions of 2021, as selected by our Chief Editors. The concentration of the silver nitrate staining solution is determined by the gel thickness. 2022, Article ID 6243971, 9 pages, 2022. https://doi.org/10.1155/2022/6243971, 1Department of Biomedical Engineering, Shenzhen Peoples Hospital (The Second Clinical Medical College, Jinan University, The First Affiliated Hospital, Southern University of Science and Technology), Shenzhen, 518020 Guangdong, China. Silver staining is the colorimetric approach that detects total protein with the highest sensitivity. Next, an image scale recognition software Image J (https://imagej.nih.gov/ij/) was used to perform image recognition and statistics of particle size. Depending upon the amount of silver incorporated into the protein bands, a different color of the gel is produced on silver staining. The image on the left is the result of the original method before optimization, and the image on the right is the result of the method after optimization. As a result, native proteins are more reactive in silver staining than unfolded proteins produced by SDS. Controlling the specificity and effectiveness of silver ion binding to proteins, as well as the successful conversion (development) of bound silver to metallic silver, necessitates the use of a variety of sensitizer and enhancer chemicals which can cause chemical crosslinking of the proteins in the gel matrix, limiting compatibility with destaining and elution methods for analysis by mass spectrometry (MS). For a gel with a thickness of 0.53mm, a 0.1% concentration of silver nitrate is ideal, and larger concentrations should be utilized for ultrathin gels to compensate for diffusion through thin gels during formation. Silver staining is an effective approach for detecting proteins separated by SDS-PAGE because of its efficiency in detecting proteins present in nanograms.
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