A silver staining procedure for nucleic acids in polyacrylamide gels without fixation and pretreatment

Silver staining of nucleic acid has been widely used in molecular marker analysis such as simple sequence repeat (SSR), single-strand conformation polymorphism (SSCP), and amplified fragment-length polymorphism (AFLP). Many alternatives to silver staining methods have been described, but these methods are not efficient or cost-effective. Here we report a silver staining method that requires less than 10 min for one gel and can save chemicals as well. It has a detection limit of approximately 5.6 pg of DNA/mm² in nondenaturing polyacrylamide gels and 12.8 pg/mm² in denaturing polyacrylamide gels.     

Detection of polyhydroxyalkanoate synthase activity on a polyacrylamide gel

This study presents a method to detect active polyhydroxyalkanoate (PHA) synthase on a polyacrylamide gel that combines the polyhydroxybutyrate (PHB) polymerization reaction with Sudan Black B staining. After separation of the protein samples on a modified sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the slab gel was submerged in a buffer containing β-hydroxybutyryl-coenzyme A (3-HBCoA) as substrate and incubated at room temperature for in vitro PHB polymerization. The active PHA synthase catalyzed 3-HBCoA into the PHB polymer and was stained with Sudan Black B. The active PHA synthase appeared as a dark blue band. The activity staining was of high sensitivity, capable of detecting 3.9 ng (0.273 mU) of Cupriavidus necator H16 PHA synthase purified from recombinant Escherichia coli. The detection sensitivity of activity staining was comparable to that of Western blotting analysis. Furthermore, the high sensitivity of activity staining enabled specific detection of the active PHA synthase in the crude extract of wild-type strain C. necator H16. This study provides a rapid, sensitive, and highly specific method for detecting active PHA synthase in gel. The method could be applied to detecting PHA synthase from wild-type bacteria and to the process of enzyme purification.     

Direct detection of rutin-degrading isozymes with polyacrylamide gel electrophoresis

Rutin-degrading enzymes (RDEs) specifically hydrolyze the glycosidic linkages of rutin, producing quercetin and rutinose. Here we report a reliable and sensitive polyacrylamide gel electrophoresis and staining method for the detection of RDE isozymes, which is based on the aqueous solubility difference between rutin and quercetin, as well as the ultraviolet absorbance of quercetin. With this novel method, we achieved a detection limit of 12 ng with 107 U of RDE activity, enabling us to detect at least five RDE isozymes in tartary buckwheat seeds.     

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

Silver staining is very sensitive for detection of proteins in polyacrylamide gels and different procedures have been published. By combining and modifying some of the recipes, a very reproducible method, which is based upon staining with diamine complexes of silver, has been developed. The background staining is negligible and reduced silver does not precipitate on the gel surface. The technique works very well for sodium dodecyl sulfate-polyacrylamide gel electrophoresis in both homogeneous and in gradient gels as well as for two-dimensional (2-D) PAGE. It was possible to detect 1-10 ng of protein corresponding to approximately 50 pg/mm2, provided that a discontinuous buffer system was used, which gives sharp bands.     

Ultrasensitive staining of nucleic acids with silver

A method for ultrasensitive detection of proteins on polyacrylamide gels by staining with silver, recently described by C. R. Merril, D. Goldman, S. A. Sedman, and M. H. Ebert (Science211, 1437–1438 (1981)), was applied with slight modifications to staining nucleic acids. Silver staining of double-stranded DNA was at least 100 times as sensitive as fluorescence staining with ethidium bromide, and at least 20 times as sensitive as staining with ammoniacal silver. The limit of detection of double-stranded DNA was approximately 25–50 pg/band with a cross-sectional area of 5 mm². The intensities of silver staining of double-stranded fragments 271 bp or longer from HaeIII endonuclease digests of φX174 RF DNA were linear over a concentration range of 0.25 to 4 ng DNA/band. RNA and single-stranded DNA species as short as 10 to 20 nucleotides were detected with high sensitivity after electrophoresis on denaturing gels containing urea, suggesting that silver staining may be applicable to the sequencing of a few micrograms of unlabeled DNA. Methods for staining DNA using ammoniacal silver were relatively insensitive for small DNA fragments.     

Determination of bicyclol in dog plasma using liquid chromatography–tandem mass spectrometry

A sensitive and accurate method for determination of bicyclol in dog plasma was developed. Thermo Scientific TSQ Quantum triple quadrupole system with multiple ion monitoring (MIM) positive scanning mode was applied. Bicyclol and DDB (IS) sodium adduct molecular ions were monitored at m/z 413 and m/z 441 in both Q1 and Q3, respectively. The collision energy in Q2 was set to 15 eV. Precipitation method was employed in the extraction of bicyclol and DDB from the biological matrix. The method was validated over 1–500 ng/mL for bicyclol. The recovery was 96.5–109.5%, and the limit of quantitation (LOQ) detection was 1 ng/mL for bicyclol. The intra- and inter-day precision of the method at three concentrations was 3.3–14.3% with accuracy of 99.9–109.0%. The method was successfully applied to bioequivalence studies of bicyclol controlled-release formulation to obtain the pharmacokinetic parameters.     

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

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

Silver staining of native and denatured eucaryotic DNA in agarose gels

A modified method of silver staining for native and denatured eucaryotic DNA in 1% agarose gel is described. This method is at least fivefold more sensitive than ethidium bromide staining, with a detection limit of 2.5 ng for total DNA. The calibration curve is linear within the range 5-30 ng of single-stranded and double-stranded DNA. This method is especially advantageous for electrophoretic assessment of DNA molecular weights.     

Sensitive and rapid quantification of the cannabinoid receptor agonist naphthalen-1-yl-(1-pentylindol-3-yl)methanone (JWH-018) in human serum by liquid chromatography–tandem mass spectrometry

The current paper describes a validated method for the detection and quantification of naphthalen-1-yl-(1-pentylindol-3-yl)methanone (JWH-018), an ingredient of a herbal mixture called “Spice”, by means of HPLC–ESI–MS–MS in serum. Lower limit of detection and lower limit of quantification were 0.07 and 0.21 ng/ml, respectively. In 2 subjects who consumed ca. 50 μg/kg of JWH-018 by smoking, the active ingredient was detected by means of the described method. Thereby, the serum concentrations reached values of approx. 10 ng/ml and dropped within 3 h very fast (<10% of the measured maximum concentrations).     

Quantitative analysis of lignocaine and metabolites in equine urine and plasma by liquid chromatography–tandem mass spectrometry

In this paper, a method for the sensitive and reproducible analysis of lignocaine and its four principal metabolites, monoethylxylidide (MEGX), glycylxylidide (GX), 3-hydroxylignocaine (3-HO-LIG), 4-hydroxylignocaine (4-HO-LIG) in equine urine and plasma samples is presented. The method uses liquid chromatography coupled to tandem mass spectrometry operating in electrospray ionisation positive ion mode (+ESI) via multiple reaction monitoring (MRM). Sample preparation involved solid-phase extraction using a mixed-mode phase. The internal standard adopted was lignocaine-d₁₀. Lignocaine and its metabolites were successfully resolved using an octadecylsilica reversed-phase column using a gradient mobile phase of acetonitrile and 0.1% (v/v) aqueous formic acid at a flow rate of 300 μL/min. Target analytes and the internal standard were determined by using the following transitions; lignocaine, 235.2 > 86.1; 3-HO-LIG and 4-HO-LIG, 251.2 > 86.1; MEGX, 207.1 > 58.1; GX, 179.1 > 122.1; and lignocaine-d₁₀, 245.2 > 96.1. Calibration curves were generated over the range 1–100 ng/mL for plasma samples and 1–1000 ng/mL for urine samples. The method was validated for instrument linearity, repeatability and detection limit (IDL), method linearity, repeatability, detection limit (MDL), quantitation limit (LOQ) and recovery. The method was successfully used to analyse both plasma and urine samples following a subcutaneous administration of lignocaine to a thoroughbred horse.     

Sensitive detection of C-reactive protein in serum by immunoprecipitation–microchip capillary gel electrophoresis

Sepsis represents a significant cause of mortality in intensive care units. Early diagnosis of sepsis is essential to increase the survival rate of patients. Among others, C-reactive protein (CRP) is commonly used as a sepsis marker. In this work we introduce immune precipitation combined with microchip capillary gel electrophoresis (IP–MCGE) for the detection and quantification of CRP in serum samples. First high-abundance proteins (HSA, IgG) are removed from serum samples using affinity spin cartridges, and then the remaining proteins are labeled with a fluorescence dye and incubated with an anti-CRP antibody, and the antigen/antibody complex is precipitated with protein G-coated magnetic beads. After precipitation the complex is eluted from the beads and loaded onto the MCGE system. CRP could be reliably detected and quantified, with a detection limit of 25 ng/μl in serum samples and 126 pg/μl in matrix-free samples. The overall sensitivity (LOQ = 75 ng/μl, R² = 0.9668) of the method is lower than that of some specially developed methods (e.g., immune radiometric assay) but is comparable to those of clinically accepted ELISA methods. The straightforward sample preparation (not prone to mistakes), reduced sample and reagent volumes (including the antibodies), and high throughput (10 samples/3 h) are advantages and therefore IP–MCGE bears potential for point-of-care diagnosis.     

A simple and sensitive HPLC fluorescence method for determination of tadalafil in mouse plasma

A simple and sensitive high-performance liquid chromatographic (HPLC) method utilizing fluorescence detection was developed for the determination of the phosphodiesterase type 5 inhibitor tadalafil in mouse plasma. This method utilizes a simple sample preparation (protein precipitation) with high recovery of tadalafil (∼98%), which eliminates the need for an internal standard. For constituent separation, the method utilized a monolithic C₁₈ column and a flow rate of 1.0 mL/min with a mobile phase gradient consisting of aqueous trifluoroacetic acid (0.1% TFA in deionized water pH 2.2, v/v) and acetonitrile. The method calibration was linear for tadalafil in mouse plasma from 100 to 2000 ng/mL (r > 0.999) with a detection limit of approximately 40 ng/mL. Component fluorescence detection was achieved using an excitation wavelength of 275 nm with monitoring of the emission wavelength at 335 nm. The intra-day and inter-day precision (relative standard deviation, RSD) values for tadalafil in mouse plasma were less than 14%, and the accuracy (percent error) was within −14% of the nominal concentration. The method was utilized on mouse plasma samples from research evaluating the potential cardioprotective effects of tadalafil on mouse heart tissue exposed to doxorubicin, a chemotherapeutic drug with reported cardiotoxic effects.     

Quantitation of tetramethylene disulfotetramine in human urine using isotope dilution gas chromatography mass spectrometry (GC/MS and GC/MS/MS)

Tetramethylene disulfotetramine (tetramine) is a rodenticide associated with numerous poisonings was extracted and quantified in human urine using both gas chromatography/mass spectrometry (GC/MS) and GC/tandem mass spectrometry (MS/MS). 1200 μL samples were prepared using a ¹³C₄-labeled internal standard, a 96-well format, and a polydivinyl-benzene solid phase extraction sorbent bed. Relative extraction recovery was greater than 80% at 100 ng/mL. Following extraction, samples were preconcentrated by evaporation at 60 °C, and reconstituted in 50 μL acetonitrile. One-microliter was injected in a splitless mode on both instruments similarly equipped with 30 m × 0.25 mm × 25 μm, 5% phenyl-methylpolysiloxane gas chromatography columns. A quantification ion and a confirmation ion (GC/MS) or analogous selected reaction monitoring transitions (GC/MS/MS) were integrated for all reported results. The method was characterized for precision (5.92–13.4%) and accuracy (96.4–111%) using tetramine-enriched human urine pools between 5 and 250 ng/mL. The method limit of detection was calculated to be 2.34 and 3.87 ng/mL for GC/MS and GC/MS/MS, respectively. A reference range of 100 unexposed human urine samples was analyzed for potential endogenous interferences on both instruments—none were detected. Based on previous literature values for tetramine poisonings, this urinary method should be suitable for measuring low, moderate, and severe tetramine exposures.     

Determination of lipoic acid in human plasma by HPLC-ECD using liquid–liquid and solid-phase extraction: Method development,validation and optimization of experimental parameters

A rapid, inexpensive, sensitive and specific HPLC-ECD method for the determination of lipoic acid in human plasma was developed and validated over the linearity range of 0.001–10 μg/ml using naproxen sodium as an internal standard (IS). Extraction of lipoic acid and IS from plasma (250 μl) was carried out with a simple one step liquid–liquid extraction using dichloromethane. Similarly solid-phase extraction was carried out using dichloromethane as extraction solvent. The separated organic layer was dried under the stream of nitrogen at 40 °C and the residue was reconstituted with the mobile phase. Complete separation of both lipoic acid and IS at 30 °C on Discovery HS C18 RP column (250 mm × 4.6 mm, 5 μm) was achieved in 6 min using 0.05 M phosphate buffer (pH 2.5 adjusted with phosphoric acid):acetonitrile (50:50, v/v) as a mobile phase pumped at the rate of 1.5 ml/min using electrochemical detector in DC mode at the detector potential of 1.0 V. The limit of detection and limit of quantification of lipoic acid were 200 pg/ml and 1 ng/ml, respectively. While on column limit of detection and limit of quantification of lipoic acid were 10 and 50 pg/ml, respectively. The absolute recoveries of lipoic acid with liquid–liquid and solid-phase extraction were 98.43, 95.65, 101.45, and 97.36, 102.73, 100.17% at 0.5, 1 and 5 μg/ml levels, respectively. Coefficient of variations for both intra-day and inter-day were between 0.28 and 4.97%. The method is validated and will be quite suitable for the analysis of lipoic acid in the plasma of human volunteers as well as patients with diabetes and cardiovascular diseases.     

Development and validation of an HPLC–FLD method for milbemectin quantification in dog plasma

Milbemectin is a widely used veterinary antiparasitic agent. A high-performance liquid chromatography with fluorescent detection (HPLC–FLD) method is described for the determination of milbemectin in dog plasma. The derivative procedure included mixing 1-methylimizole [MI, MI-ACN (1:1, v/v), 100 μL], trifluoroacetic anhydride [TFAA, TFAA-ACN (1:2, v/v), 150 μL] with a subsequent incubation for 3 s at the room temperature to obtain a fluorescent derivative, which is reproducible in different blood samples and the derivatives proved to be stable for at least 80 h at room temperature. HPLC method was developed on C18 column with FLD detection at an excitation wavelength of 365 nm and emission wavelength of 475 nm, with the mobile phase consisting of methanol and water in the ratio of 98:2 (v/v). The assay lower limit of quantification was 1 ng/mL. The calibration curve was linear over concentration range of 1–200 ng/mL. The intra- and inter-day accuracy was >94% and precision expressed as % coefficient of variation was <5%. This method is specific, simple, accurate, precise and easily adaptable to measure milbemycin in blood of other animals.     

A signal-amplified electrochemical immunosensor for aflatoxin B1 determination in rice

A sensitive and simple electrochemical immunosensor based on enzymatic silver deposition amplification was constructed for the detection of aflatoxin B₁ (AFB₁) in rice. The immunosensor was based on an indirect competitive format between free AFB₁ and aflatoxin B₁-bovine serum albumin (AFB₁-BSA) conjugate immobilized on the electrode surface for binding to a fixed amount of anti-AFB₁ antibody. Then the alkaline phosphatase (ALP)-labeled anti-mouse immunoglobulin G (IgG) secondary antibody was bound to the electrode surface through reaction with primary antibody. Finally, ALP catalyzed the substrate, ascorbic acid 2-phosphate, into ascorbic acid that reduced silver ions in solution to metal silver deposited onto the electrode surface. Linear sweep voltammetry was carried out to quantify the metal silver, which indirectly reflected the amount of the analyte. The experimental parameters, such as the dilution ratio of antibody and the concentration of AFB₁-BSA conjugate, have been evaluated and optimized. At the optimal conditions, the working range of the electrochemical immunosensor was from 0.1 to 10 ng/ml with a detection limit of 0.06 ng/ml. Good recoveries were obtained for the detection of spiked rice samples. So, the proposed method in this article could find a good use for screening AFB₁ in real samples.     

An LC-MS/MS method for determination of forsythiaside in rat plasma and application to a pharmacokinetic study

A highly sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed for the determination of forsythiaside in rat plasma using epicatechin as internal standard. The analytes were extracted by solid-phase extraction and chromatographied on a C₁₈ column eluted with a gradient mobile phase of acetonitrile and water both containing 0.2% formic acid. The detection was performed by negative ion electrospray ionization in multiple reaction monitoring mode, monitoring the transitions m/z 623 → 161 and m/z 289 → 109 for forsythiaside and epicatechin, respectively. The assay was linear over the concentration ranges of 2.0–50.0 and 50.0–5000.0 ng/mL with limits of detection and quantification of 0.2 and 1.0 ng/mL, respectively. The precision was <10.8% and the accuracy was >91.9%, and extraction recovery ranged from 81.3% to 85.0%. This method was successfully applied to a pharmacokinetic study of forsythiaside in rats after intravenous (20 mg/kg) and oral (100 mg/kg) administration, and the result showed that the compound was poorly absorbed with an absolute bioavailability being approximately 0.5%.     

Silver staining for carboxymethylated metallothioneins in polyacrylamide gels

A sensitive method for detecting metallothioneins (MTs) by use of silver staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of carboxymethylated MTs was developed. Carboxymethylation of metallothioneins is indispensable because it prevents their aggregation, thereby allowing each of them to be detected as a single band by SDS-PAGE. However, when the gel was subjected to the silver-staining method of C. R. Merril, D. Goldman, S. A. Sedman, and M. H. Ebert [(1981) Science 211, 1437-1438], the image of carboxymethylated purified MTs was totally negative. Pretreatment of the gel with 1% sodium thiosulfate just prior to the silver-staining procedure successfully reversed the negative image of carboxymethylated MTs. Further, they could be detected with a limit of nanogram levels per lane. This method can be applied to MTs in cell extracts from cultured cell lines treated with cadmium or to those from liver of cadmium-intoxicated mice.     

A simple and cost-effective solid-phase protein nano-assay using polyacrylamide-coated glass plates

A new solid-phase protein nano-assay is suggested for simple and sensitive estimation of protein content in sample buffers (a 1-μl sample is sufficient for analysis). The assay is different from conventional “on-filter” assays in that it uses inexpensive fully transparent polyacrylamide gel (PAAG)-coated glass plates as solid support and, thus, combines the convenience of “on-membrane” staining with the sensitivity and ease of documentation of “in-gel” staining (and, therefore, is especially suited for standard lab gel documentation systems). The PAAG plates assay is compatible with all dyes for in-gel protein staining. Depending on the sensitivity of the staining protocol, the assay can be used in macro-, micro-, and nano-assay formats. We also describe a low-cost two-component colloidal Coomassie brilliant blue G-250 (CBB G-250) staining protocol for fast quantitative visualization of proteins spotted on a PAAG plate (the detection limit is up to 2 ng of proteins even when using a Nikon CoolPix digital camera and white light transilluminator instead of a gel scanner). The suggested colloidal CBB G-250 protocol could also be used for visualizing nano-amounts of proteins in polyacrylamide gels. The PAAG plate assay could be useful for proteomic applications and, in general, for all cases where a fast, sensitive, and easily documentable cost-effective solid-phase protein assay is required.     

Application of one-step liquid chromatography–electrospray tandem MS/MS and collision-induced dissociation to quantification of ezetimibe and identification of its glucuronated metabolite in human serum: A pharmacokinetic study

A new one-step liquid chromatography–electrospray tandem MS/MS method is described to quantify ezetimibe (EZM) a novel lipid lowering drug in human serum. Also using collision-induced dissociation (CID) of the analyte, identification and chromatographic separation of its major metabolite, ezetimibe glucuronide (EZM-G) is achieved in this study. A thawed serum aliquot of 100 μL was deproteinated by addition of 500 μL methanol containing omeprazole as internal standard (I.S.). Separation of the drug, its metabolite and the I.S. were achieved using acetonitrile–water (70:30, v/v) as mobile phase at flow rate of 0.5 mL/min on a MZ PerfectSil target C18 column. Multiple reaction monitoring (MRM) mode of precursor–product ion transition (408.7 → 272.0 for EZM and 345 → 194.5 for the I.S.) was applied for detection and quantification of the drug while, EZM-G was chromatographically separated and identified using CID. The analytical method was linear over the concentration range of 1–32 ng/mL of EZM in human serum with a limit of quantification of 1 ng/mL. The coefficient variation values of both inter- and intra-day analysis were less than 8% whereas the percentage error was less than 3.7. The validated method was applied in a randomized cross-over bioequivalence study of two different EZM preparations in 24 healthy volunteers.