A protocol for stripping and reprobing of Western blots originally developed with colorimetric substrate TMB

Western blotting is a widely used analytical technique for detection of specific protein(s) in a given sample of tissue/cell homogenate or extract. Both chemiluminescence (CL) and colorimetric detections can be used for imaging Western blots. Colorimetric substrates offer background free, sensitive, and clean imaging results directly on the blotted membrane and provides more accurate profile with respect to prestained marker. However, blots stained with colorimetric substrates cannot be reused since no stripping protocols have been reported for such blots, thus limiting their reuse for detection of another protein. In the present study, for the first time, we report a novel method of stripping Western blots developed with the colorimetric substrate TMB for detection of a low‐abundant protein and reprobing of these blots after stripping for detection of a more abundant protein through CL procedure. The stripping procedure utilizes a stripping buffer consisting of β‐mercaptoethanol, SDS, and Tris‐HCl and a washing buffer consisting of PBS added with 0.1% Tween‐20 involves a series of steps and facilitates accurate detection of the second protein (i.e., more abundant protein) in the stripped blot through CL. The protocol is reproducible and facilitates saving of precious clinical samples, in addition to saving cost and time as compared to the existing procedures.     

BirA*-protein A fusion protein based BioEnhancer amplifies western blot immunosignal

Western blot (protein immunoblot) is a widely used analytical technique in molecular biology. Utilizing the specific recognizing primary antibody, proteins immobilized on various matrix are investigated by subsequent visualization steps, for example, by the horse radish peroxidase conjugated secondary antibody incubation. Methods to improve the sensitivity in protein identification or quantification are appreciated by biochemists. Herein, we report a new strategy to amplify Western blot signals by constructing a probe with proximal labeling and IgG targeting abilities. The R118G mutation attenuated the biotin-AMP binding affinity of the bacterial biotin ligase BirA*, offering a proximity-dependent labeling ability, which could be used as a signal amplifier. We built a BirA*-protein A fusion protein (BioEnhancer) that specifically binds to IgG and adds biotin tags to its proximal amine groups, enhancing the immunosignal of target proteins. In our experiments, the BioEnhancer system amplified the immunosignal by tenfold compared to the standard western blot. Additionally, our strategy could couple with other signal enhancement methods to further increase the western blot sensitivity.     

Spreader-CDR system for efficient,reproducible, and frugal western blot

Efficient antibody incubation is a vital step for successful western blot. During the incubation, a thin antibody-depleted layer is created around the blotting membrane, which limits antibody binding. Although the conventional batch shaking method is ineffective against it, this layer can be easily disrupted by cyclic draining and replenishing (CDR) of the antibody solution during membrane incubation. Previously, we introduced a closed and rotating cylindrical chamber as a tool to implement CDR for western blots (rCDR). A new open bucket-style chamber was devised for easier operation and the possibility of process automation. Instead of rotation as in rCDR, rocking it back and forth achieved the CDR antibody incubation (R-CDR). The chamber was then equipped with a spreader-rod to facilitate the uniform movement of the antibody solution across the membrane surface. Hence, it was named spreader CDR (S-CDR). Compared to the batch incubation method, both the S-CDR and R-CDR devices produced significantly enhanced signals and developed faster results. There were several additional benefits of using the spreader-rod, which included uniform antibody binding across the membrane, reduced usage of antibodies, and the ability to recover results even from mishandled, creased membranes. The S-CDR device ensures better blots and can be easily implemented in existing western blot protocols.     

A novel method that improves sensitivity of protein detection in PAGE and Western blot

We have developed a simple and inexpensive method that improves sensitivity of protein and antigen detection in standard PAGE procedures. Our technique uses a sample microloader device with a funnel‐like structure, filled with a 4% stacking gel. When attach to the top of a polyacrylamide slab gel, the proteins in a sample are concentrated by electrophoresis into a small volume as they emerge from the device's narrow outlet. Our microloader has several advantages over previous devices, including simple assembly, high versatility, and absence of cross‐contamination between lanes. Addition of this device to a slab gel results in a fivefold increase in the sensitivity of antigen detection in a Western blot. As a result, less protein is required for loading and signal detection. Our protocol is a straightforward modification of a standard experimental technique, and is especially useful when only limited sample quantities are available.     

Western blot normalization: Time to choose a proper loading control seriously

Recent research has questioned the validity of housekeeping proteins in Western blot. Our present study proposed new ideas for Western blot normalization that improved the reproducibility of scientific research. We used the Gene Expression Omnibus (GEO) database and the web tool GEO2R to exclude unstable housekeeping genes quickly. In ischemic heart tissues, actin and tubulin changed significantly, whereas no statistically significant changes were observed in the expression of genes relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Besides, the reliability of GAPDH was further examined by Western blot. Additionally, unstable housekeeping genes were found in other animal models of cardiovascular medicine. We also found that sodium dodecyl sulfate and temperature significantly impacted the results of Ponceau S staining. Membranes stained with Ponceau S after immunodetection could avoid this interference, and the coefficients of variation for post-immunodetection staining are lower than those produced by GAPDH immunodetection. Overall, we described a new use of differential gene expression analysis and proposed a modified Ponceau S staining method, which provided researchers with a proper loading control for Western blot and hence could improve reproducibility in research.     

Characterization of a biopharmaceutical protein and evaluation of its purification process using automated capillary Western blot

This paper describes the application of an automated size‐based capillary Western blot system (Sally instrument) from ProteinSimple, Inc., for biopharmaceutical fusion‐Fc protein characterization and evaluation of its purification process. The fusion‐Fc protein column purification from an excess of single chain Fc polypeptide and removal of an enzyme coexpressed for protein maturation have been demonstrated using an automated capillary Western system. The clearance of a selected host cell protein (HCP) present in cell culture of fusion‐Fc protein was also quantitatively monitored throughout the protein purification process. Additionally, the low levels of fusion‐Fc product‐related impurities detected by traditional slab gel Western blot were confirmed by the automated capillary Western system. Compared to the manual approach, the automated capillary Western blot provides the advantages of ease of operation, higher sample throughput, greater linearity range, and higher precision for protein quantitation.     

Fractionation of the human plasma proteome for monoclonal antibody proteomics‐based biomarker discovery 2: Antigen identification by dot‐blot array screening

Immunization with complex mixtures, like the human plasma resulted in the generation of cloned mAb libraries (PlasmaScan? and QuantiPlasma? libraries, with >1000 individual mAbs) reacting with a nonredundant set of antigenic epitopes. mAb proteomics refers to quasi‐hypothesis‐free profiling of plasma samples with nascent or cloned mAb libraries for the discovery of disease‐specific biomarkers. Once mAbs with biomarker potential have been identified, the next task is the determination of cognate antigens recognized by the respective mAbs. To determine the cognate protein antigen corresponding to each individual mAbs in the cloned mAb libraries, we have separated human plasma by consecutive steps of desalting and various chromatography procedures. The process resulted in 783 fractions, which we termed “Analyte Library” (AL). The AL represents the human plasma proteome in relatively low‐protein complexity fractions. Here, to determine the utility of the AL, we selected ten plasma proteins and checked for their presence in the fractions. Among the ten cases, the distribution of four selected plasma proteins matched expectations, as these proteins were present only in a few fractions corresponding to their physical, chemical, and biochemical properties. However, in six cases, we observed “smear” ‐like distribution or complete absence of the proteins, suggesting that protein–protein interactions or protein variants may alter the observed plasma distribution profiles. Nevertheless, we conclude that the AL is an efficient, high throughput tool to complement the mAb biomarker discovery process with cognate protein antigen identification for each mAbs.     

Comprehensive quantitative analysis by spark source mass spectrometry: A technique on the brink

Spark Source Mass Spectrometry has been shown to be a particularly sensitive method for the detection of small concentrations of a wide range of elements in solid samples. However, unless improved commercial instrumentation becomes available it is a method which, in spite of its great potential, will fall into progressive decline.     

Qualitative and quantitative analysis of iridoid glycosides in the flower buds of Lonicera species by capillary high performance liquid chromatography coupled with mass spectrometric detector

A highly sensitive and specific method, based on capillary high performances liquid chromatography coupled with single quadrupole mass spectrometry using electrospray ionization (capillary HPLC-ESI/MS), is proposed for the identification and quantification of iridoid glycosides in the flower buds of five Lonicera species. A Zorbax SB-C18 (0.3 mm × 150 mm, 5 μm) capillary column and a gradient elution with methanol-acetonitrile-aqueous acetate acid were utilized. The most intensive electrospray ionisation signals were found in the negative ion spectra owing to CH₃COO⁻ adducts. Eight iridoid glycosides derived from the flower buds of Lonicera species were analyzed by mass spectrometry: sweroside (IG1), 7-O-ethyl sweroside (IG2), 7-epi vogeloside (IG3), secoxyloganin (IG4), secoxyloganin 7-butyl ester (IG5), dimethyl-secologanoside (IG6), centauroside (IG7), and loganin (IG8) using combined information on retention time, the molecular ion mass and fragment ion masses. Detection limits were lower than 1.9 ng/mL in selected ion monitoring (SIM) mode and all calibration curves showed good linear regression (r² > 0.9938) within test ranges. The validated method was successfully applied to analyze eight iridoid glycosides in the flower buds of five Lonicera species and provided a new basis of assessment on quality of Flos Lonicerae.     

CPRM: a new method for quantitative detection of peroxide formation during oxygen reduction using the rotating ring-disc electrode technique

A new procedure to detect peroxide formation quantitatively during oxygen reduction using the rotating ring-disc electrode (RRDE) technique is described. The new procedure is called the Cyclic Potential Ring Measurement (CPRM) method. In this technique, the ring electrode is continuously cycled between 850 mV and 1600 mV versus RHE at a sweep rate of 500 . Concurrently, the disc electrode is stepped potentiostatically in the potential region of oxygen reduction. For oxygen reduction on gold, the CPRM technique indicates 100% peroxide formation in the first wave region whereas the conventional RRDE technique indicated only 20 to 30% peroxide formation. The continuous sweeping of the ring electrode in the CPRM technique regenerates continuously a fresh, active ring electrode surface that is less susceptible to interference from low levels of impurities in the solution. Consequently, the CPRM technique provides a more stable, reproducible ring surface for peroxide detection than the conventional technique.To insure that the potential sweeping of the ring does not affect its response adversely, we measured the electrode collection coefficient with a model system (Fe(CN)³⁻₆/Fe(CN)⁴⁻₆), in which low levels of solution impurities would not interfere. The collection coefficient measured using the CPRM and conventional techniques with the model system agreed within ±2.4%.     

The end-on mechanism for lattice filling: Comparison with the conventional mechanism and application to the car-parking problem

We present the exact solution for the sequential, random, irreversible filling of one-dimensional lattices by linear n-mers using the end-on filling mechanism. The results are extrapolated to then limit (a variation on the car-parking problem) to yield a saturation coverage (packing density) of 0.7350. The end-on filling mechanism involves two steps for a single filling event. First, the landing site for one endpoint of the filling species is chosen and then the second endpoint is subsequently chosen (fromunfilled sites an appropriate distance from the first endpoint). We compare this mechanism to the conventional, one-step filling mechanism, where both endpoints of the filling species are chosen simultaneously. We present results detailing how the lattice saturation coverage varies for the two mechanisms. In addition, we extend our analysis to consider filling in the presence of a time-dependent, random distribution of inactive sites.     

Qualitative and quantitative determination of the major coumarins in Zushima by high performance liquid chromatography with diode array detector and mass spectrometry

A method, high-performance liquid chromatography coupled with diode array and electrospray tandem mass spectrometry (HPLC-DAD-ESI-MS), was developed to qualitatively identify and quantitatively determine the 10 major active coumarins of Zushima. The analysis was performed by using a ZORBAX SB-C18 analytical column (250 mm x 4.6 mm ID, 5 microm) at gradient elution of 0.5% aqueous formic acid and acetonitrile with diode array detection (325 nm). The method was validated for linearity, precision, accuracy, limit of detection and quantification. The proposed method was successfully applied for the qualitative and quantitative analysis of 10 coumarins in five different species of Zushima which had great variation on the contents of investigated coumarins.     

Investigation of the continuous flow of the sample solution on the performance of electromembrane extraction: Comparison with conventional procedure

In this study, electromembrane extraction from a flowing sample solution, termed as continuous‐flow electromembrane extraction, was developed and compared with conventional procedures for the determination of four basic drugs in real samples. Experimental parameters affecting the extraction efficiency were further studied and optimized. Under optimum conditions, linearity of continuous‐flow procedure was within 8.0–500 ng/mL, while it was wider for conventional procedures (2.0–500 ng/mL). Moreover, repeatability (percentage relative standard deviation) was found to range between 5.6 and 10.4% (n  = 3) for the continuous‐flow procedure, with a better repeatability than that of conventional procedures (2.3–5.5% (n  = 3)). Also, for the continuous‐flow procedure, the estimated detection limit (signal‐to‐noise ratio = 3) was less than 2.4 ng/mL and extraction recoveries were within 8–10%, while the corresponding figures for conventional procedures were less than 0.6 ng/mL and 42–60%, respectively. Thus, the results showed that both continuous flow and conventional procedures were applicable for the extraction of model compounds. However, the conventional procedure was more convenient to use, and thus it was applied to determine sample drugs in real urine and wastewater samples.     

A comprehensive NMR methodology to assess the composition of biobased and biodegradable polymers in contact with food

A nuclear magnetic resonance (NMR) methodology was assessed regarding the identification and quantification of additives in three types of polylactide (PLA) intended as food contact materials. Additives were identified using the LNE/NMR database which clusters NMR datasets on more than 130 substances authorized by European Regulation No. 10/2011. Of the 12 additives spiked in the three types of PLA pellets, 10 were rapidly identified by the database and correlated with spectral comparison. The levels of the 12 additives were estimated using quantitative NMR combined with graphical computation. A comparison with chromatographic methods tended to prove the sensitivity of NMR by demonstrating an analytical difference of less than 15%. Our results therefore demonstrated the efficiency of the proposed NMR methodology for rapid assessment of the composition of PLA.     

A kinetic study of the copolymerization of maleic anhydride with ethyl vinyl ether using the rotating-sector technique

The results of polymerization and initiation experiments at 25°C show that the alternating copolymerization of maleic anhydride (monomer 1) and ethyl vinyl ether (monomer 2) is consistent with a theoretical mechanism defined by the usual rate constants (in L/mol s): Propagation: (k_p)₁₁ = 0.00; (k_p)₁₂ = 1.66 × 10⁵, (k_p)₂₂ = 2.0 × 10³; and (k_p)₂₁ = 2.04 × 10⁵. Termination (k_t)₁₁ = 7.40 × 10¹⁰; (k_t)₁₂ = 52.8 × 10¹⁰; (k_t)₂₂ = 1.33 × 10¹⁰. The relative magnitudes of the two cross-propagation constants and the three termination constants are consistent with accepted theory with regard to polarity, resonance, and steric factors. The steady-state and rotating-sector experiments were carried out in a dilatometer using azobisdiisobutyronitrile as the initiator, acetone as the solvent, and UV light of 365 nm wave length.     

Laser-induced breakdown spectroscopy of archaeological findings with calibration-free inverse method: Comparison with classical laser-induced breakdown spectroscopy and conventional techniques

A modified version of the calibration-free (CF) method was applied to the analysis of a set of archaeological brooches made of various copper-based alloys and coming from the archaeological site of Egnatia (Apulia, Southern Italy). The developed methodology consists in determining the plasma temperature by reversing the set of equations employed in the usual CF algorithm, and it is thus referred to as “inverse method”. The plasma temperature is determined for one certified standard, by using its known elemental composition as an input data, and then applied to the set of unknown samples to evaluate their composition in a CF mode. The feasibility of such an approach is demonstrated by comparing the results obtained with classical LIBS (drawing calibration lines with a series of matrix-matched certified standards) and with independent measurements performed with a conventional technique (LA-ICP-MS).     

Comparison of localization and delocalization indices obtained with Hartree-Fock and conventional correlated methods: effect of Coulomb correlation

Atomic populations and localization [lambda(A)] and delocalization [delta(A,B)] indices (LIs and DIs) are calculated for a large set of molecules at the Hartree-Fock (HF), MP2, MP4(SDQ), CISD, and QCISD levels with the 6-311++G(2d,2p) basis set. The HF method and the conventional correlation methods [MP2, MP4(SDQ), CISD, and QCISD] yield distinct sets of LIs and DIs. Yet, within the four conventional correlation methods the differences in atomic populations and LIs and DIs are small. Relative to HF, the conventional correlation methods [MP2, MP4(SDQ), CISD, QCISD] yield virtually the same LIs and DIs for molecules with large charge separations while LIs and DIs that differ significantly from the HF values--the LIs are increased and DIs decreased--are obtained for bonds with no or small charge separations. Such is the case in the archetypal homopolar molecules HC(triple bond)CH, H2C=CH2, CH3-CH3, and "protonated cyclopropane" C(3)H(7) (+), in which case the bonding may be atypical. Relative to HF, the typical effect of the conventional correlation methods is to decrease the DI between atoms.     

Simultaneous determination of peroxydisulfate and conventional inorganic anions by ion chromatography with the column‐switching technique

The application of ion chromatography with the column‐switching technique for the simultaneous analysis of peroxydisulfate and conventional inorganic anions in a single run is described. With this method, conventional inorganic anions were separated by consecutive elution through both the guard column and separation column, but peroxydisulfate that only passed through the guard column had a good peak shape and short retention time. A series of standard solutions consisting of target anions of various concentrations from 0.01 to 75 mg/L were analyzed, with a correlation coefficient (r) ≥ 0.9990. The limits of detection were in the range of 0.49–9.84 μg/L based on the S/N of 3 and a 25 μL injection volume. RSDs for retention time, peak area, and peak height were all <1.77%. A spiking study was performed with satisfactory recoveries between 97.6 and 103.4% for all anions. The quantitative determination of peroxydisulfate and conventional inorganic anions in surface waters was accomplished within 18 min by this column‐switching technique.     

Determination of phosphorus using capillary electrophoresis and micro-high-performance liquid chromatography hyphenated with inductively coupled plasma mass spectrometry for the quantification of nucleotides

We performed the quantification of phosphorus in deoxynucleotides using capillary electrophoresis (CE) and micro-HPLC (μHPLC) hyphenated with inductively coupled plasma mass spectrometry (ICP-MS). DNA and its component units have conventionally been determined by photometry; however, more selective and sensitive methods are needed for small biological samples. CE and μHPLC offer the advantages of good separation and small consumption of samples, and ICP-MS is a highly sensitive technique for the determination of a chemical element. Therefore, we have developed an interface device for combining CE and μHPLC with ICP-MS for quantifying nucleotides based on phosphorus content. The interface utilizes 4.5 μL/min for nebulizing and effective introduction of the sample into ICP. The samples of nucleotides and free phosphoric acid were well separated in the CE–ICP-MS measurement, and the calibration curves (1–100 μg/mL) of the nucleotides showed a linear (R² > 0.999) increase in intensity. Similarly, the samples of nucleotides were baseline separated using μHPLC–ICP-MS, and the calibration curves of the nucleotides were linear (R² > 0.998). The detection limits of these species and phosphorus in nucleotides using CE–ICP-MS and μHPLC–ICP-MS were 0.77–6.5 ng/mL and 4.0–6.5 ng/mL, respectively. These values were about one or two orders lower than those in a previous report. The sample volumes of these experiments were calculated to be about 10 nL and 50 nL per analysis. Therefore, these analytical methods have the potential to be useful for the determination of biological samples, such as DNA and RNA molecules.