Protein Staining Method Research Articles

Microwave-assisted and methanol/acetic acid-free method for rapid staining of proteins in SDS-PAGE gels

We describe a microwave-assisted, methanol and acetic acid-free, inexpensive method for rapid staining of SDS-PAGE proteins. Only citric acid, benzoic acid, and Coomassie brilliant blue G-250 (CBG) were used. Microwave irradiation reduced the detection duration, and proteins in a clear background were visualized within 30 min of destaining, after 2 min of fixing and 12 min of staining. By using this protocol, comparable band intensities were obtained to the conventional methanol/acetic acid method.

No-stain protein labeling as a potential normalization marker for small extracellular vesicle proteins

Western blot analysis of relative protein expression relies on appropriate reference proteins for data normalization. Small extracellular vesicles (sEVs), or exosomes, are increasingly recognized as potential indicators of the physiological state of cells due to their protein composition. Therefore, accurate relative sEVs protein quantification is crucial for disease detection and prognosis applications. Currently, no documented ubiquitous reference proteins are identified for precise normalization of a protein of interest in sEVs. Here we showed the use of total protein staining method for sEVs protein normalization in western blots of samples where conventional housekeeping proteins like β-actin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) are not always detected in the sEVs western blots. The No-Stain™ Protein Labeling (NSPL) method showed high sensitivity in sEVs-protein labeling and facilitated quantitative evaluation of changes in the expression pattern of the protein of interest. Further, to show the robustness of NSPL for expression analysis, the results were compared with quantitative mass spectroscopy analysis results. Here, we outline a comprehensive method for protein normalization in sEVs that will increase the value of protein expression study of therapeutically significant sEVs.

Improvements for Tissue-Chopping-Based Immunofluorescence Staining Method of Chloroplast Proteins

Immunofluorescence staining is a very common method for the subcellular localization study of proteins. A tissue-chopping-based immunofluorescence staining method for chloroplast proteins overcomes the restriction of plant cell wall, makes the operation simpler, and uses less experimental materials. Here we provide some improvements for this method. We found that the stained tissues can be directly observed with a confocal microscope without tissue lysis. Samples maintained at a low temperature (0-4 °C) throughout the process can reduce the intensity of chlorophyll autofluorescence and the background signal. A low temperature is also good for the storage of the sample. Fluorescence signal of the stained samples can be kept for several weeks if they are stored at -20 °C. FtsZ is an essential component of the chloroplast division apparatus. We demonstrated this method with the immunofluorescence staining of FtsZ1 in wildtype Arabidopsis and some chloroplast division mutants. We also successfully tested this method by the immunofluorescence staining of FtsZ1 in many other plants, including woody plants. With these procedures, the performance of tissue-chopping-based immunofluorescence staining method are further improved.

Modification in Silver Staining Procedure for Enhanced Protein Staining.

Silver staining is an excellent technique for detecting proteins that are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). 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. The present study was conducted to enhance the detection ability of the protein staining method. 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. 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 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, 0.5% AMP, 0.5% PVP, 0.5% Tween-80 reagents significantly influenced the morphology, size, potential, and dispersion of silver ions. These results suggested a new idea for further improving the detection ability of protein silver staining.

A rapid method for protein staining in polyacrylamide gels using water saturated with chloroform.

Polyacrylamide gel electrophoresis, followed by an appropriate staining, is a popular and useful analytical procedure for protein identification and characterization. The aim of this study was to develop a method for protein visualization in polyacrylamide gels that would be alternative to Coomassie blue or silver staining. The proposed method is simple, fast and inexpensive. The optimized protocol for protein staining and visualization takes as little as 6 minutes and utilizes deionized water and chloroform. Fluorescence of proteins is induced by UV light and can be detected with a standard transilluminator.

Differential Proteome Analysis Using 2D-DIGE.

Classical 2D-PAGE allows comparison and quantitation of proteomes by visualization of protein patterns using gel stains and comparative image analysis. The introduction of fluorescent reagents for protein labeling (difference in-gel electrophoresis or DIGE) has brought substantial improvement in this field. It provides multiplexing of up to three samples in one gel, higher sensitivity compared to normal protein staining methods, and a higher linear range for quantitation. This article gives detailed protocols for 2D-DIGE, including both minimal and saturation labeling.

A sensitive and reversible staining of proteins on blot membranes

A modified, sensitive and reversible method for protein staining on nitrocellulose (NC) and polyvinylidine fluoride (PVDF) membranes was developed in Western blotting. The method employed Congo red staining to visualize proteins on different blot membranes. Staining of proteins with Congo red dye is more faster procedures. According to the experimental results, approximate 20 ng proteins could be detected in 3 min in room temperature. The staining on the proteins is easily reversible with Congo red destaining solution for NC and PVDF membranes, so that the blot membranes can be reused for Western blotting. In addition, we confirmed that the staining method is fully compatible with Western blot detection. NC and PVDF membranes treatment with Congo red staining does not interfere with conventional chemiluminescent substrates of peroxidase. As compared to MemCode reversible protein stain kits from Pirece Biotechnology, the staining technique is more sensitive, lower of cost, convenient and not adversely affecting subsequent Western blotting results. On the other hand, the stain is more sensitive than the Ponceau S staining. Therefore, Congo red staining is a promising and ideal alternative for current protein stain. Besides, the binding modes of Congo red or Ponceau S stain were investigated using various 2D and 3D molecular docking and demonstrated potential molecular basis for sensitivity of Congo red staining are higher than Ponceau S.

A Single-Step Simultaneous Protein Staining Procedure for Polyacrylamide Gels and Nitrocellulose Membranes byAlta During Western Blot Analysis.

A simple method for staining of proteins simultaneously on sodium dodecyl sulfate (SDS)-polyacrylamide gels and nitrocellulose membranes by Alta during Western blot analysis is described. A 5% solution of Alta, a commercially available cosmetic preparation, is added in the upper tank buffer during electrophoresis. On completion of electrophoresis, the gel is washed in distilled water, viewed on a white light plate and a transilluminator to photograph the protein profiles. The gel is processed for Western blot transfer of proteins onto a nitrocellulose membrane, and upon completion, the protein profiles on the membrane are viewed and photographed as stated above. The membrane can then be processed for immunostaining as per the standard procedure. Thus, the staining procedure using Alta is simple, rapid (without any need of destaining) and cost-effective.

Counterion Dye Staining of Proteins in One- and Two-Dimensional Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis and Tryptic Gel Digestion of Stained Protein for Mass Spectrometry.

A fast and matrix-assisted laser desorption/ionization-mass spectrometry compatible protein staining method in one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis is described. It is based on the counterion dye staining method that employs oppositely charged two dyes, zincon and ethyl violet to form an ion-pair complex. The protocol including fixing, staining, and quick washing steps can be completed in 1-1.5h depending upon gel thickness. It has the sensitivity comparable to the colloidal Coomassie Brilliant Blue G stain using phosphoric acid as a component of staining solution (4-8ng). The counterion dye stain does not induce protein modifications that complicate interpretation of peptide mapping data from mass spectrometry. Considering the speed, sensitivity, and compatibility with mass spectrometry, the counterion dye stain may be more practical than any other dye-based protein stains for routine proteomic researches.

An ultrasensitive stain for negative protein detection in SDS-PAGE via 4′,5′-Dibromofluorescein

A highly sensitive method for brief and economical staining of proteins in 1-D and 2-D sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) by 4′,5′-Dibromofluorescein (DBF) was developed in this study. Down to 0.025–0.05ng protein could be detected within 10min (only 2 steps) by DBF stain, which is approximately 10-fold more sensitive than those of Eosin Y (EY) and SYPRO Ruby stains, and 20-fold more sensitive than that of imidazole-zinc (IZ) negative stain. In addition, the LC-MS/MS results indicated that the newly developed staining method is compatible with the downstream protein identification. Moreover, the mechanism of DBF stain was investigated by molecular docking.

A method for rapid and sensitive negative staining of proteins in SDS-PAGE using 2',7'-dichlorofluorescein.

We report here a rapid and sensitive technique for negative visualization of protein in 1D and 2D SDS-PAGE by using 2', 7'-dichlorofluorescein (DCF), which appeared as transparent and colorless bands in an opaque gel matrix background. For DCF stain, down to 0.1-0.2ng protein could be easily visualized within 7min by only two steps, and the staining is fourfold more sensitive than that of Eosin Y (EY) negative stain and glutaraldehyde (GA) silver stain, and eightfold more sensitive than that of the commonly used imidazole-zinc (IZ) negative stain. Furthermore, DCF stain provided good reproducibility, linearity, and MS compatibility compared with those of IZ stain. In addition, the potential staining mechanism was investigated by colorimetric experiment and molecular docking, and the results demonstrated that the interaction between DCF and protein occurs mainly via van der waals force, electrostatic interaction, and hydrogen bonding.

An improved immunofluorescence staining method for chloroplast proteins.

An improved immunofluorescence staining method significantly facilitates the visualization of the subcellular localization of interested proteins in chloroplasts. As an important technical approach, immunofluorescence staining is widely used in the subcellular localization study of interested proteins. During the study of the functions of chloroplast division proteins, immunofluorescence staining was frequently adopted. Previously, a method has been developed to study the localization of a chloroplast division protein, FtsZ. However, it is laborious and time-consuming. In this study, we report a modified immunofluorescence staining method, in which protoplasts were isolated from leaf tissues, and then fixed for immunofluorescence staining. The time of the experiment was significantly reduced to several hours. Furthermore, we used correction pen in the fixation procedure and a new way to coat the slide, which greatly saved the cost of the experiment. With the chloroplast division protein ARC6 as an example, we can get a good fluorescence signal. Moreover, the localization of ARC6 in two chloroplast division mutants, arc3 and arc5, and three other plant species, such as cabbage, radish and pea, was also successfully analyzed with our new method. Overall, the immunofluorescence staining method we reported here is very practical, and it significantly facilitates the visualization of the subcellular localization of interested proteins in plant cells.

Induced Differentiation of Epithelioid Carcinoma Cell Lines: Evidence for Tumor Cell Quantal Mitosis

The effects of growth factors and calcium concentrations present in different culture media on induction of terminal differentiation were investigated for four different epidermoid carcinoma cell lines, Hela, KB, A431, and SCC-25, and their responses determined relative to those elicited by normal human keratinocytes subjected to these culture conditions. Differentiation status was determined cyto-chemically by a validated keratin protein staining method, and by autoradiographic analyses. Growth and differentiation promoting factors that influenced the direction of integrated control of growth and differentiation in normal human keratinocytes were found to be effective for some cell lines but not others. The factors examined were 1) high density arrest in serum-free and serum-containing media, 2) media shifts from high density culture in serum-containing media to low density growth factor-depleted or supplemented serum-free medium, and 3) the concentration of calcium in the media. The extent and degree of differentiation achieved varied among different cell lines depend on the presence or absence of serum, EGF and insulin protein growth factors. Certain growth media appear to sponsor keratin protein, cyto-chemically-detected differentiation, and evidence of quantal mitotic division in low density HeLa cell and SCC25 cell cultures. Epidermoid carcinoma cell lines retain limited capacity to commit to early stages of cell differentiation.

Sodium polyanethol sulfonate (SPS) falsifies protein staining and quantification and how to solve this problem

Sodium polyanethol sulfonate (SPS) is an anionic detergent with a broad range of activities and applications. While studying the excretion of cytoplasmic proteins in Staphylococcus aureus SPS was used as cell lysis inhibitor. When investigating the protein pattern of culture supernatants from cells grown in the absence or presence of SPS by Coomassie blue stained polyacrylamide gel the amount of protein bands was significantly decreased in the presence of SPS, suggesting that this effect was due to inhibition of cell lysis. However, various control studies showed that the apparent decreased protein secretion was an artifact due to the interference of SPS with Coomassie blue- and silver-staining. The only alternative method that was uninfluenced by SPS was imidazole–SDS–zinc staining. This is the method of choice particularly when protein interfering compounds are present in the extracts. For protein quantification in liquid samples the bicinchoninic acid (BCA) assay appeared to be the method of choice in the presence of SPS. The assay is based on neutral peptide bonds and is therefore rather insensitive to interfering compounds. This study shows that SPS and most likely also related detergents might falsify conventional protein staining and quantification methods.

Protein Stains to Detect Antigen on Membranes.

Western blotting (protein blotting/electroblotting) is the gold standard in the analysis of complex protein mixtures. Electroblotting drives protein molecules from a polyacrylamide (or less commonly, of an agarose) gel to the surface of a binding membrane, thereby facilitating an increased availability of the sites with affinity for both general and specific protein reagents. The analysis of these complex protein mixtures is achieved by the detection of specific protein bands on a membrane, which in turn is made possible by the visualization of protein bands either by chemical staining or by reaction with an antibody of a conjugated ligand. Chemical methods employ staining with organic dyes, metal chelates, autoradiography, fluorescent dyes, complexing with silver, or prelabeling with fluorophores. All of these methods have differing sensitivities and quantitative determinations vary significantly. This review will describe the various protein staining methods applied to membranes after western blotting. "Detection" precedes and is a prerequisite to obtaining qualitative and quantitative data on the proteins in a sample, as much as to comparing the protein composition of different samples. "Detection" is often synonymous to staining, i.e., the reversible or irreversible binding by the proteins of a colored organic or inorganic chemical.

Comparative Histopathological Diagnosis of Cutaneous Melanoma by H&E, Special Staining and Immunohistochemical Methods against Cutaneous Squamous Cell Carcinoma in Horse and Bovine

Background: The aim of this study was to compare the role of special staining and to test melanoma associated antigens against paraffin embedded bovine and equine melanomas in order to assess their ability and utility as diagnostic tools for diagnosis and confirmation of the melanomas. Methods: From 1991-2012, 13 cases were reviewed histologically, and each case was found to contain a large focus of melanoma and squamous cell carcinoma (SCC) on the hematoxylin and eosin–stained tissue section. All cases had documented cutaneous melanoma and SCC. Serial 5 μm-thick sections were cut from routine formalinfixed paraffin blocks of 4 case cutaneous SCC (including 2 cases from horses and 2 cases from cattle) and 9 case cutaneous melanomas (including 6 horses cases and 3 cattle cases) from the files of the Department of Pathology have been diagnosed by H&E staining method. For diagnosis confirmation, Fontana-masson silver method, melanin bleaching method and immunohistochemical staining for S-100 protein and HMB45 antigen were applied. Majority of cases encompassed intradermal histologic pattern. Results: In 6 cases out of 9 tumors, both S-100 protein and HMB45 staining methods were positive, whereas there were suspicious results for 3 cases. In all cases, results of Fontana-masson silver and melanin bleaching methods were positive. In 4 cases of squamous cell carcinoma, results of 3 tests were negative. Diagnosis of suspicious melanoma tumor cases must be confirmed or reconfirmed by special staining methods and/or immunohistochemical method. Conclusions: The standard of practice in diagnosing melanoma is to use a panel of antibodies consisting primarily of S-100 protein and HMB-45. For spindle cell and desmoplastic melanomas, however, S-100 protein and HMB-45 should remain within antibody panel since S-100 protein is most sensitive marker for these entities and evidence is lacking that either Melan-A or Mitf are better markers than HMB-45 for recognizing spindle cell melanocytic lesions.

Establish a flow cytometric method for quantitative detection of Beclin-1 expression

Flow cytometry is an advanced technology for efficient, rapid, specific and multi-parameter analysis of single cells in various basic research fields including cytobiology, immunology, genetic, hematology and other basic research. Beclin-1 protein is an important indicator in monitoring autophagic activity. However, quantitative flow cytometry had been rarely reported till now to be applied in the detection of Beclin-1 expression. The present study was aimed to establish a flow cytometric method for quantitative detection of Beclin-1 expression by employing the autophagy inhibitor 3-methyladenine as the control. A multi-parameter optimal method for Beclin-1 protein staining is as follows. 2% bovine serum albumin in phosphate buffered saline was used for sample block. Concentration of primary antibody was 0.004μg/μL. Samples were incubated at room temperature (25°C) for 30min. The prepared samples had better to be detected immediately or to be stored at 4°C and detected within 6h, otherwise the samples should be fixed in 1% paraformaldehyde storing at 4°C and detected within 3 d. Furthermore, we employed the immunohistochemistry to validate the method in vivo, the results confirmed flow cytometric method. The established flow cytometric analysis for Beclin-1 protein has the advantage of simpleness, speediness, sensitivity and reproducibility.

Synthesis and Cytotoxicity of Chalcones and 5‐Deoxyflavonoids

Chalcones 1~8 and 5-deoxyflavonoids 9~22 were synthesized in good yields by aldol condensation, Algar-Flynn-Oyamada reaction, glycosidation, and deacetylation reaction, respectively, starting from 2-acetyl phenols substituted by methoxy or methoxymethoxy group and appropriately benzaldehydes substituted by methoxy, methoxymethoxy group, or chlorine. Among them, 13 and 17~22 are new compounds. The cytotoxicity bioassays of these chalcones and 5-deoxyflavonoids were screened using the sulforhodamine B (SRB) protein staining method, and the results showed that compounds 2, 4, 5, 6, 10, 15, and 19 exhibited moderate cytotoxicity against the cancer cell line of MDA-MB-231, U251, BGC-823, and B16 in comparison with control drugs (HCPT, Vincristine, and Taxol).

Pig sperm membrane microdomains contain a highly glycosylated 15–25-kDa wheat germ agglutinin-binding protein

A highly glycosylated protein, which has unique, novel features in localization, structure, and potential function, is found in pig sperm, and named WGA-gp due to its high binding property with wheat germ agglutinin (WGA). WGA-gp is localized mainly in flagella and enriched in membrane microdomains or lipid rafts. It is not detected by ordinary protein staining methods due to a high content of both N- and O-glycans consisting of neutral monosaccharides. Interestingly, WGA-gp may be involved in intracellular Ca2+ regulation. Treatment of sperm with anti-WGA-gp antibody enhances the amplitude of Ca2+ oscillation without changing the basal intracellular Ca2+ concentrations. All these features of WGA-gp, except for different carbohydrate structures occupying most part of the molecules, are similar to those of flagellasialin in sea urchin sperm, which regulates the intracellular Ca2+ concentration. Presence of carbohydrate-enriched flagellar proteins involved in intracellular Ca2+ regulation may be a common feature among animal sperm.

Analysis of oxidative stress-induced protein carbonylation using fluorescent hydrazides

Protein carbonyl detection has been commonly used to analyze the degree of damage to proteins under oxidative stress conditions. Most laboratories rely on derivatization of carbonyl groups with dinitrophenylhydrazine followed by Western blot analysis using antibodies against the dinitrophenyl moiety. This paper describes a protein carbonyl detection method based on fluorescent Bodipy, Cy3 and Cy5 hydrazides. Using this approach, Western blot and immunodetection are no longer needed, shortening the procedure and increasing accuracy. Combination of Cy3 and Cy5 hydrazides allows multiplexing analyses in a single two-dimensional gel. Derivatization with Bodipy hydrazide allows easy matching of the spots of interest and those obtained by general fluorescent protein staining methods, which facilitates excising target proteins from the gels and identifying them. This method is effective for detecting protein carbonylation in samples of proteins submitted to metal-catalyzed oxidation "in vitro" and assessing the effect of hydrogen peroxide and chronological aging on protein oxidative damage in yeast cells.