Fast fluorescent staining of protein in sodium dodecyl sulfate polyacrylamide gels by palmatine

A fast and sensitive protein fluorescent detection method in SDS-PAGE using the natural product palmatine is described. Palmatine is an alkaloid found in various plants exhibiting a broad spectrum of antibiotic activity in humans. The sensitivity of palmatine staining is similar to those of the SYPRO Red, SYPRO Tangerine, and SYPRO Orange protein gel stains - about 4 ng per protein band. This detection sensitivity is comparable to colloidal CBB staining. Since proteins stained with palmatine do not need destaining, the staining procedure can be easily shortened and completed in about 30 min. Stained proteins can be photographed using a UV transilluminator. The results of the present study suggest that the palmatine staining is sensitive, rapid, low cost, and safe for a broad application to the research of protein.     

A rapid and simplified method for protein silver staining in polyacrylamide gels

Silver staining is widely used to detect protein in polyacrylamide gels when high sensitivity is required. A simple and rapid protocol for silver staining of proteins following PAGE was developed in the present study. The number of steps was reduced compared to conventional protocol by combining fixing, rinsing, and soaking into a single impregnating step, thus achieving detection of proteins in 20 min. The present method is as sensitive as current protocols with the advantage of saving time and costs.     

Highly selective fluorescence turn-on determination of fluoride ions via chromogenic aggregation of a silyloxy-functionalized salicylaldehyde azine

A novel fluorescent chemsensor TBS-protected salicylaldehyde azine (TSAA) for fluoride ion was developed based on aggregation-induced emission (AIE). The probe TSAA was prepared by the reaction of salicylaldehyde azine (SAA) with tert-butyldimethylsilyl chloride (TBS-Cl) via an unusual synthetic methodology and shows only non-emission. Upon treatment with fluoride in aqueous MeCN solution, the TBS protective group of probe TSAA was removed readily and the fluorescence of the probe was switched on, which resulted in a new fluorescence peak around 543 nm. The fluorescent intensity at 543 nm increases linearly with fluoride ion concentration in the range 1–50 μmol L^?1. This proposed probe shows excellent selectivity toward fluoride ion over other common anions and cations.     

A statistical comparison of silver and SYPRO Ruby staining for proteomic analysis

Silver staining has been the method most commonly employed for high sensitivity staining of proteins following two-dimensional gel electrophoresis. Whilst this method offers detection in the nanogram range it does have major drawbacks including a lack of linearity, nonstoichiometric staining of proteins, a lack of compatibility with the microchemical preparation of proteins for identification by mass spectrometric techniques, and a highly subjective assessment of the staining endpoint. SYPRO Ruby is a relatively new, ruthenium complex-based stain which is reported to offer advantages over silver, particularly in overcoming the limitations cited above. We describe a series of experiments where several protein staining procedures commonly employed are compared. To enable optimization of the in situ digestion procedure, a statistical approach has been undertaken. The effects of a variety of staining, digestion, and analysis protocols on the downstream processing of a test radiolabeled protein were studied. The data confirms that as well as offering sensitivity similar to silver, SYPRO Ruby staining is reproducible, linear, and offers a higher level of compatibility with the identification of proteins by mass spectrometry.     

An optimal method of DNA silver staining in polyacrylamide gels

A silver staining technique has widely been used to detect DNA fragments with high sensitivity on polyacrylamide gels. The conventional procedure of the silver staining is tedious, which takes about 40-60 min and needs five or six kinds of chemicals and four kinds of solutions. Although our previous improved method reduced several steps, it still needed six kinds of chemicals. The objective of this study was to improve further the existing procedures and develop an optimal method for DNA silver staining on polyacrylamide gels. The novel procedure could be completed with only four chemicals and two solutions within 20 min. The steps of ethanol, acetic acid, and nitric acid precession before silver impregnation have been eliminated and the minimal AgNO3 dose has been used in this up-to-date method. The polyacrylamide gel of the DNA silver staining displayed a golden yellow and transparent background with high sensitivity. The minimum 0.44 and 3.5 ng of DNA amount could be detected in denaturing and nondenaturing polyacrylamide gel, respectively. This result indicated that our optimal method can save time and cost, and still keep a high sensitivity for DNA staining in polyacrylamide gels.     

Improved conditions for silver–ammonia staining of DNA in polyacrylamide gel

An improved silver–ammonia staining method for DNA on polyacrylamide gels is described. In this method, staining of DNA using silver–ammonia complex allows high sensitivity, low cost, low toxicity, and simple protocol without requiring fixation and sensitization steps. The protocol takes less than 40 min to complete, with a detection limit of 1.5 pg of single DNA band on polyacrylamide gels, approximately 30‐fold higher than that of original silver–ammonia staining method. Furthermore, this novel technique not only exhibits high sensitivity for large DNA fragment, but also shows a better trend to detect low‐base‐pair DNA compared with other silver staining methods.     

An expanding negative detection method for the visualization of DNA in polyacrylamide gels by eosin Y

A sensitive and easy technique has been developed for the negative detection of DNA following PAGE using eosin Y. After electrophoresis, gels are fixed and stained within 40 min to provide a detection limit of 0.1-0.2 ng of single DNA band, which appears as transparent and colorless under the opaque gel matrix background. The sensitivity of the new stain is fourfold better than zinc-imidazole negative and ethidium bromide stains. Furthermore, the newly developed staining method has been successfully applied to RNA visualization in polyacrylamide gels. In addition, the inclusion of inorganic salts in staining solution was also investigated, which has great effect on the staining efficiency.     

Efficient and sensitive method of DNA silver staining in polyacrylamide gels

DNA silver staining is widely used to detect DNA fragment in polyacrylamide gel with high sensitivity. Conventional procedures of the silver staining involve several steps, which take about 40 min to 2 h in total. To improve the efficiency of DNA silver staining, a more efficient protocol is developed in this study. The procedure comprises only four steps including impregnating, rinsing, developing, and stopping, and could be completed within 20 min. Nitric acid and ethanol in the silver-impregnation step of the new procedure eliminates the need for prior treatment of gels with a fixing solution and following rinse prior to impregnation with silver. The procedure has high sensitivity and long storage lifetime. The minimum detectable mass of DNA is 0.44 and 3.5 ng in denaturing and nondenaturing polyacrylamide gel, respectively.     

An optimal method of DNA silver staining in polyacrylamide gels

DNA silver staining has widely been used to detect DNA fragments in polyacrylamide gels with high sensitivity. We developed an optimal method for DNA silver staining on polyacrylamide gels. The novel procedure can be completed within 10 min instead of over 20 min with the conventional methods. The sensitivity is significantly improved by the silver-ion sensitizer (Eriochrome black T (EBT)) and the minimum of 0.11 and 1.75 ng of DNA amount can be detected in denaturing and nondenaturing polyacrylamide gel, respectively. Compared with the conventional silver staining methods, the improved optimal method can save time and display high sensitivity, color uniformity, and long storage time of the staining gels.     

Background-free,fast protein staining in sodium dodecyl sulfate polyacrylamide gel using counterion dyes,zincon and ethyl violet

A background-free, fast protein staining method in polyacrylamide gel electrophoresis using an acidic dye, zincon (ZC) and a basic dye, ethyl violet (EV) is described. It is based on the counterion dye staining technique that employs two oppositely charged dyes to form an ion-pair complex in staining solution. The selective binding of free dye molecules to proteins in acidic solution produces bluish violet-colored bands. It is a rapid and end-point staining procedure, involving only fixing and staining steps that are completed in 1-1.5 h. The detection limit of this method is 8-15 ng of protein that is comparable to the sensitivity of the colloidal Coomassie Brilliant Blue G (CBBG) stain. Due to its sensitivity and speed, this stain may be more practical than any other dye-based stains for routine laboratory purposes.     

Detection of glutathione reductase after electrophoresis on native or sodium dodecyl sulfate polyacrylamide gels

Commercial glutathione reductase (GR) from spinach and yeast (Saccharomyces cerevisiae) were stained on 7.5% native polyacrylamide gel electrophoresis (PAGE) gels or 15% sodium dodecyl sulfate (SDS)-PAGE gels with or without further purification by a 2',5'-ADP Sepharose 4B affinity column. For SDS-PAGE gels, the SDS was removed first by washing twice with 25% isopropanol in 10 mM Tris-HCl (pH 7.9) for 10 min. The gel was then dipped in a 50 mM Tris-HCl buffer (pH 7.9) containing 4.0 mM oxidized glutathione (GSSG), 1.5 mM NADPH, and 2 mM 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) for 20 min. The GR activity was negatively stained in the dark by a solution containing 1.2 mM 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 1.6 mM phenazine methosulfate (PMS) for 5-10 min. The contrast between the clear zone of GR activity and the purple background was found in both native and SDS-PAGE gels. This negative staining method can detect GR as little as 0.064 units and 0.0032 units, respectively, for spinach and yeast sources. Under reduced SDS-PAGE gels, the GR activity band located on 72 kDa for spinach and 51 kDa for yeast. This fast and sensitive method could be used during enzyme purification and for characterization of GR from different sources under different physiological stages or conditions.     

High-efficiency protein extraction from polyacrylamide gels for molecular mass measurement by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry

A simple and fast method of protein extraction from Coomassie Brilliant Blue (CBB)-stained polyacrylamide gels suited for molecular mass measurement of proteins by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) is reported. Proteins in CBB-stained gel pieces were extracted by a 10-min soaking in 0.1 M NaOH at 25 degrees C. The recovery of this one-step extraction method was 34-73% for proteins <67 kDa. CBB adduction to proteins during mass spectrometric analysis was avoided by a destaining step before the alkaline extraction. The molecular mass values of the extracted proteins coincided with those of purified proteins within +/-0.01-0.10% deviation for all the proteins <36 kDa. Because of the high extraction recovery, mass measurement was possible for the proteins extracted from CBB-stained gels with loaded protein quantities as little as 34 ng for cytochrome c, alpha-lactalbumin, myoglobin, beta-lactoglobulin, trypsinogen, and carbonic anhydrase (12.4-29.0 kDa), 340 ng for glyceraldehyde-3-phosphate dehydrogenase (35.6 kDa) and albumin (66.3 kDa). This method provides a highly efficient approach to utilize CBB-stained one- or two-dimensional gels for whole protein analysis using MALDI-TOF-MS.     

A rapid and effective method for silver staining of PCR products separated in polyacrylamide gels

With the development of molecular quantitative genetics, particularly, genetic linkage map construction, quantitative trait loci mapping or genes fine mapping and association analysis etc., more and more PCR products separated in polyacrylamide gels need to be silver‐stained. However, conventional silver‐staining procedures are complicated and time‐consuming as they require a lot of preparation and handling of several solutions prior to use. In this study, a simple and rapid protocol for silver staining of PCR products was developed. The number of steps was reduced compared to conventional protocols, thus achieving detection of PCR products in 7 min, saving time and resources. Fixation and staining solution and developing solution in present staining procedure allowed a reutilization for 12 and 8 times, respectively, reducing the cost greatly. Meanwhile, the sensitivity was significantly improved with the improved method and the minimum of 0.097 ng/μL of DNA amount can be detected in denaturing polyacrylamide gel. The protocol developed in this study will facilitate the development of molecular quantitative genetics.     

Detection of PEGylated proteins in polyacrylamide gels by reverse staining with zinc and imidazole salts

The reverse staining, with imidazole-SDS-zinc, of PEG-linked proteins separated by SDS-PAGE was studied. Using model conjugates (interferon-alpha 2b (IFN-alpha2b) reacted with either a branched-chain (40,000) PEG (PEG2,40) or a linear monomethoxy PEG polymer (Mr of 12,000) and chromatographically purified monoPEG2,40-IFN-alpha2b), conventional small-format analytical gels (<1 mm thick) showed typical detection patterns (i.e., transparent, colorless bands clearly discernible against a zinc imidazolate-generated white gel background), in less than 20 min. Nonreacted (free) PEG was almost undetected, as expected. The reverse-stained PEGylated IFN-alpha2b patterns were qualitatively indistinguishable from those of parallel gels stained with iodine (I2). The LOD was estimated in the low nanogram range (e.g., at about 7 ng for mono- or bi-PEG2,40 IFN-alpha2b per lane on gradient (4-17%) gels). Also, this stain allowed the visualization of Coomassie blue-undetected PEG-IFN bands, and could be restained with I2. PEGylated species of lysozyme, a low-molecular-weight peptide, ovalbumin, and chymotrypsin were used to demonstrate the generality of this stain. We also show (i) how to counteract the adverse effect of some parameters (e.g., gel thickness above 1 mm, long gel length, low (e.g., 4-6%) acrylamide concentration) on the reverse staining process and (ii) that the properties of the reverse-stained PEGylated proteins remain unchanged, as judged by analyzing both the ion exchange chromatography-based positional isomer separation profile and enzyme-linked immunosorbent response of PEG-IFN recovered from gels. Consequently, this technique may be useful for the rapid analysis or the small-scale preparation of PEGylated proteins.     

Band broadening of DNA fragments isolated by polyacrylamide gel electrophoresis in capillary electrophoresis

Polyacrylamide gel electrophoresis (PAGE) is used frequently for isolation and purification of DNA fragments. In the present study, DNA fragments extracted from polyacrylamide gels showed significant band broadening in capillary electrophoresis (CE). A pHY300PLK (a shuttle vector functioning in Escherichia coli and Bacillus subtilis) marker, which contained nine fragments ranging from 80 to 4870 bp, was separated by PAGE, and each fragment was isolated by phenol/chloroform extraction and ethanol precipitation. After extraction from the polyacrylamide gel, the peaks of the isolated DNA fragments exhibited band broadening in CE, where a linear poly(ethylene oxide) was used as a sieving matrix. The theoretical plate numbers of the DNA fragments contained in the pHY300PLK marker were >10⁶ for all the fragments before extraction. However, the DNA fragments extracted from the polyacrylamide gel showed decreased theoretical plate numbers (5–20 times smaller). The degradation of the theoretical plate number was significant for middle sizes of the DNA fragments ranging from 489 to 1360 bp, whereas the largest and smallest fragments (80 and 4870 bp) had no obvious influence. The band broadening was attributed to contamination of the DNA fragments by polyacrylamide fibers during the separation and extraction process.     

Polyacrylamide gel electrophoresis of proteoglycans in large-pore gradient gels

A procedure is described for preparing large-pore polyacrylamide gradient gels (1.2–16% T, 10–4% C) that allow the separation of large proteoglycans of molecular mass up to several millions.     

Separation performance of single-stranded DNA electrophoresis in photopolymerized cross-linked polyacrylamide gels

Considerable effort has been directed toward optimizing performance and maximizing throughput in ssDNA electrophoresis because it is a critical analytical step in a variety of genomic assays. Ultimately, it would be desirable to quantitatively determine the achievable level of separation resolution directly from measurements of fundamental physical properties associated with the gel matrix rather than by the trial and error process often employed. Unfortunately, this predictive capability is currently lacking, due in large part to the need for a more detailed understanding of the fundamental parameters governing separation performance (mobility, diffusion, and dispersion). We seek to address this issue by systematically characterizing electrophoretic mobility, diffusion, and dispersion behavior of ssDNA fragments in the 70-1,000 base range in a photopolymerized cross-linked polyacrylamide matrix using a slab gel DNA sequencer. Data are collected for gel concentrations of 6, 9, and 12%T at electric fields ranging from 15 to 40 V/cm, and resolution predictions are compared with corresponding experimentally measured values. The data exhibit a transition from behavior consistent with the Ogston model for small fragments to behavior in agreement with the biased reptation model at larger fragment sizes. Mobility data are also used to estimate the mean gel pore size and compare the predictions of several models.     

Using in situ rheology to characterize the microstructure in photopolymerized polyacrylamide gels for DNA electrophoresis

Photopolymerized cross-linked polyacrylamide hydrogels are attractive sieving matrix formulations for DNA electrophoresis owing to their rapid polymerization times and the potential to locally tailor the gel pore structure through spatial variation of illumination intensity. This capability is especially important in microfluidic systems, where photopolymerization allows gel matrices to be precisely positioned within complex microchannel networks. Separation performance is also directly related to the nanoscale gel pore structure, which is in turn strongly influenced by polymerization kinetics. Unfortunately, detailed studies of the interplay among polymerization kinetics, mechanical properties, and structural morphology are lacking in photopolymerized hydrogel systems. In this paper, we address this issue by performing a series of in situ dynamic small-amplitude oscillatory shear measurements during photopolymerization of cross-linked polyacrylamide electrophoresis gels to investigate the relationship between rheology and parameters associated with the gelation environment including UV intensity, monomer and cross-linker composition, and reaction temperature. In general, we find that the storage modulus G' increases with increasing initial monomer concentration, cross-linker concentration, and polymerization temperature. The steady-state value of G', however, exhibits a more complex dependence on UV intensity that varies with gel concentration. A simple model based on rubber elasticity theory is used to obtain estimates of the average gel pore size that are in surprisingly good agreement with corresponding data obtained from analysis of DNA electrophoretic mobility in gels cast under identical polymerization conditions.     

Capturing sodium dodecyl sulfate-treated protein antigens by antibody affinity electrophoresis

Modifications to antibody affinity electrophoresis for improved detection of proteins have been developed. The bifunctional linker glutaraldehyde is added to the polyacrylamide gel solution for better incorporation of the bait antibody into a distinct region of a 10% w/v polyacrylamide gel. The addition of glutaraldehyde alleviates the need of an electrophoresis buffer with a specific pH. The protein sample to be analyzed is treated with 2% w/v sodium dodecyl sulfate (SDS) to ensure that they carry a negative charge. The negative charge will allow the proteins to migrate towards the cathode and hence pass through the area embedded with the bait antibody. It is observed that electrophoretic migration of bovine serum albumin (BSA) or protein G ceases upon encounter with anti-BSA whereas proteins ovalbumin, beta-lactoglobulin A, and myoglobin migrate freely. However, the addition of 0.1% w/v SDS in the native gel running buffer disrupts the antibody-antigen bond and neither BSA nor protein G can be captured by anti-BSA.