protein staining techniques

The copper mesh was clamped, and the excess liquid was absorbed with filter paper. 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. Although the electric potential may not explain all the phenomena, it still reflects a certain trend for size of particles. Another element that influences the sensitivity of protein staining on gels is protein reactivity with silver ions. Detection of BSA protein using electrophoretic staining (a) Original treatment method without xylene. At present, the influence of the high-level structure of the protein on the silver staining effect cannot be determined. 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]. Yibo Jiang, LinLin Zheng, Lulin Lin, Shan Lin, Kui Xu, SiJie Deng, QiQing Zhang, "Modification in Silver Staining Procedure for Enhanced Protein Staining", BioMed Research International, vol. Few studies, however, demonstrated that color variation is due to the formation of silver chromate deposits that are incorporated into formalin-fixed proteins. The advanced phenomenon can result in the enhanced detection efficiency and sensitivity of low-concentration proteins. Read the winning articles. Compared with the original method, the sensitivity was improved after adding additives. A two-color 25-250KD protein marker was used as an electrophoresis label, followed by the gel documentation using the iBright Imaging System [1]. The combination of PVP, AMP, and Tween-80 kits resulted in more stained protein bands. However, proteins that do not contain or rarely contain cysteine residues are sometimes negatively stained [4, 5]. No related content is available yet for this article. 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. 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. The black arrow is 0.5ng target protein which is difficult to distinguish in (b) and even cannot be seen in (a). It has been observed that the larger particles are more irregular in shape, as shown in Figure 7. Relevant technical principles and methods provide a reference for further improving the detection effect and ideas of silver staining technology. Eight samples (ah) of silver ion materials: (a) 0.5% AgNO, The size of each of the 100 particles in samples (ah): (a) 0.5% AgNO. The obtained results demonstrated that the smaller the potential, the larger the particle size. The obtained results shown in Figure 5 demonstrate that adding reagents to the staining solution alone can boost the staining effect for a low concentration of the protein band. It can be performed with simple and inexpensive laboratory reagents, and the readout does not necessitate complicated and expensive equipment. 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. The statistical software SAS 12.0 was used to calculate the average value for each group. 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. Article of the Year Award: Outstanding research contributions of 2021, as selected by our Chief Editors. 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. The average value was then used as the criterion after 10 minutes of stable measurements [1315]. The copper mesh upside was put down on the sample and suspended for 1.5 minutes. SDS-PAGE of serial dilutions of BSA protein. (b) Treatment with PVP, AMP, and Tween-80 without xylene. 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 concentration of the silver nitrate staining solution is determined by the gel thickness. PVP, AMP, Tween-80 also has an effect on silver ion shape, size, potential, and dispersion. The copper mesh and filter paper were clamped and sucked up the excess liquid. 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 quantitative findings of ultratrace BSA protein in an embodiment of the present invention are shown in Figure 4. (a) Staining according to the traditional method, and (b) staining with the new method. 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. The presence of amino acid in protein structure affects the process of silver staining. Main SDS-PAGE and silver staining technique step are shown in Figure 1. 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. Following electrophoresis, a full piece of gel was obtained; however, during staining, the gel was separated and stained separately. Protein silver staining is a nonspecific staining of proteins with an explosive reaction mode. 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. As a result, native proteins are more reactive in silver staining than unfolded proteins produced by SDS. Furthermore, 0.5% AMP, 0.5% PVP, 0.5% Tween-80 reagents significantly influenced the morphology, size, potential, and dispersion of silver ions. (c) Xylene, PVP, AMP, and Tween-80 added group. The staining process sequentially consists of protein fixation, sensitization, washing, silver impregnation, and finally development of the image. Tecnai G2 Spirit TEM was used for the photo of silver ion materials and the particle scale was calibrated at 500nm at 4C [15, 18]. This work presents a new approach to further enhance the protein silver staining detection technique. The most powerful interactions occur with carboxylic acid groups (Asp and Glu), imidazoles (His), sulfhydryls (Cys), and amines (Lys). The detection sensitivity was significantly enhanced by 20200 times, allowing proteins as low as 0.1ng protein per band to be detected [1, 2]. 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]. As such, traditional staining methods have a poor effect, and the background remains high, resulting in limited detection capabilities for low-abundance proteins. 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. The color variation has been attributed to diffractive scattering by silver grains of different sizes. The authors declare that they have no conflicts of interest. Comparison of the effects of various additives. This study has been supported by funding. 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. Silver staining is an excellent technique for detecting proteins that are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Histidine was found to be the most significant amino acid for silver ion binding and efficient silver staining among all amino acids. Certain protein functional groups interact and bind with silver ions (from silver nitrate in the staining reagent). Among them, the composite treatment group 6 has the largest particle size and lower potential. Furthermore, the images also demonstrated the black spot which may be associated with the larger particle size of silver [3]. 2007, An et al. The procedure described above resulted in an increase in detection sensitivity. 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. First, 2ml of the solution was added at 25C temperature and the measurements were performed thricely. Furthermore, the background staining was decreased, which led to the enhanced detection efficiency and sensitivity of low-concentration proteins. Furthermore, the size of silver particles deposited on the protein surface has a significant impact on the protein gels color development. (b) Xylene treatment group. (a) Original method without other reagents. Moreover, Figure 3(c) shows the results obtained from the new method, employed with PVP, AMP, and Tween-80, followed by treatment with xylene. The particle size and potential difference were measured using a Malvern particle size potentiometer (ZEN 3700). Frequently, the additives resulted in an intensification of background staining. We thank the Shenzhen Science and Technology Innovation Commission for the Shenzhen Basic Research Technology Breakthrough (general project, grant number: JCYJ202103241113210027). 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 morphology and structure of the silver particles in the silver dye reagent were observed and verified by TEM. 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. 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 functional groups of proteins must be exposed in order for silver ions to attach to them. Aside from silver ions attached to proteins, silver ions in the vicinity of the protein are also required for the formation of bands. Moreover, the unclear and high background makes it difficult to detect low-concentration proteins [8]. Heukeshoven and Dernick also evaluated the silver staining of the basic homopolymers of arginine, histidine, and ornithine but not for polylysine [7]. PVP, AMP, and Tween-80 were then added. 2009). In this study, we used 0.5% of AMP, PVP, Tween-80, and xylene which led to the enhancement of the silver staining technique. The black arrow is 0.5ng target protein. 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. According to the glue observation, the proper amount of double distilled water was added to save the gel. (a) Original treatment group. 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. In Figure 2(b), the bands from left to right are different concentrations of BSA protein, i.e., 20ng, 50ng, and 100ng, and protein marker lysate. 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. The SDS-PAGE classic electrophoresis method was performed using the Beijing Liuyi electrophoresis instrument. Target protein can be checked in both of B and C groups. All reagents were made with premium-grade, which were mixed and boiled for 5 minutes before sample loading and electrophoresis. Furthermore, the detection limit was estimated to be lower than 0.5ng per protein band. A comparative evaluation of the effects of various additives has been carried out. Although the staining effect has been improved, the underlying explanation is still unknown. Silurian materials were dyed with 2% phosphotungstic acid [17], and then, 10L of the sample was dropped on a clean parafilm. Then, 10L of 2% phosphotungstic acid was dropped on the clean sealing film. 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. The dye solution on the copper mesh was suspended for 2.5 minutes. 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. 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. Silver-stained protein bands generally are dark brown or black with considerable variation in color intensity. Copyright 2022 Yibo Jiang et al. The present study was conducted to enhance the detection ability of the protein staining method. Except for the marker, the protein concentration of the band from left to right was 0.1ng, 0.2ng, 0.5ng, and 10ng. Depending upon the amount of silver incorporated into the protein bands, a different color of the gel is produced on silver staining. 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. Metallic silver is deposited onto a gels surface at the positions of protein bands in this approach. 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. According to the obtained statistics results, PVP and AMP treatment increases the size of AgNO3 particles to varying degrees. Silver staining is the colorimetric approach that detects total protein with the highest sensitivity. TEM (FEI Tecnai G2 Spirit Twin, Czech Republic) was used to observe the size and morphology of silver particles in various prepared solutions. Figure 6 shows considerable discrepancies between samples 1 and 2-8, as determined by the particle size potential analyzer ZEN 3700. Butcher and J. K. Tomkins, A comparison of silver staining methods for detecting proteins in ultrathin polyacrylamide gels on support film after isoelectric focusing,. Hence, if large amounts of silver ions are deposited on the protein bands, it makes it easier to detect low-abundance proteins. 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). 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). Several factors influence the efficiency and sensitivity of silver staining. Figure 3 shows the detection of BSA protein using electrophoretic staining. As a result, the size of the potential plays a major role in particle and silver ion deposition on the protein surface, ultimately affecting the outcome of silver staining. The samples were then subjected to ultrasonication for 10 minutes [16]. 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. 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. 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. These results suggested a new idea for further improving the detection ability of protein silver staining. All the consumables were dry at room temperature for 20 minutes. The significant correlation between the intensity of silver staining and the mole percentage of basic amino acids lysine and histidine in a protein is most likely due to the electron-donating abilities of the amino groups and imidazole groups at the termini of the lysine and histidine side chains. A protein concentration of 0.5ng in the black arrow also has been shown. 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. 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]. Other reagents can increase particle size to varying degrees. We modified the traditional staining method by adding AMP, PVP, Tween-80, and xylene to enhance the detection ability of protein staining. It is almost close to the limit of resolution. The technique is based on the simple principle that selective reduction of silver into metallic silver occurs at the initiation site in the proximity of protein molecules. TEM analyses revealed that silver ion particles in samples 28 all get larger and more irregular in shape to varying degrees compared to sample 1(control) in Figure 7. The electrophoresis bands in this experiment are BSA protein (20ng, 50ng, and 100ng) and protein marker, from left to right. This technique rapidly gained popularity owing to its high sensitivity as compared to other stains including the Coomassie Brilliant Blue R250 stains. The above-obtained results suggested that the new and improved method can effectively reduce the color rendering background, increase the color rendering brightness of the band, and improve the detection sensitivity. The potential difference when the particles aggregate can also affect silver ion aggregation [20]. Staining was observed for the various homopolymers including polymethionine, the hydrophilic basic amino acid polymers: polylysine, polyarginine, polyornithine, and polyhistidine, [6]. Lawrence, Silver-staining of proteins in polyacrylamide gels: a general overview,, L. A. In addition, the particle sizer and the potential analysis system analyzed the potential and particle size of the silver dye reagent and the size, as well as the morphology of the silver dye particles. 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 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]. 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. Herein, we used commercially available BSA (bovine serum albumin) (Sigma fatty acid-free type) standard protein samples. 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. 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. 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. Such observations have led us to develop a more versatile silver staining protocol that requires only a few stable and readily storable solutions and allows for some coordinates in terms of concentration and reaction time. The morphology and structure of the silver particles in the silver dye reagent were observed and verified by TEM transmission electron microscopy technology. 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]. (c) Treatment with PVP, AMP, and Tween-80 with xylene. In this study, the TEM results revealed that the larger the silver ion particles, the more irregular the shape, allowing for particles deposition. The result of the same batch of detection has a lower background and clearer bands. (b) The original method with the AMP group. The gel washing was carried out in double-distilled water for 5min, and then, silver staining was performed by adding 0.1% silver nitrate, 20% formaldehyde, and 0.5% xylene for 20 minutes, followed by adding 2.5% sodium carbonate and 10% formaldehyde for color development (10 minutes). The reactivity of silver ions, and hence the sensitivity of protein detection, is also affected by protein structure. To address these limitations, various changes were suggested from time to time, and hundreds of modified techniques for silver staining proteins on polyacrylamide gels are currently in use. (c) The original method with the Tween-80 groups. Next, an image scale recognition software Image J (https://imagej.nih.gov/ij/) was used to perform image recognition and statistics of particle size. When compared to the standard reference group, each treatment group had a greater particle size and a lower potential. Silver staining is an effective approach for detecting proteins separated by SDS-PAGE because of its efficiency in detecting proteins present in nanograms. The significant level dividing line was or 0.01 [13]. The left picture is the original treatment group, the middle picture is the xylene treatment group, and the right picture is the posttreatment group with xylene.

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