Band Intensity Research Articles

A one-process production of completely biotinylated proteins in a T7 expression system.

Streptavidin is a tetrameric protein with high specificity and affinity for biotin. The interaction between avidin and biotin has become a valuable tool in nanotechnology. In recent years, the site-specific biotin modification of proteins using biotin ligases, such as BirA, has attracted attention. This study established an in vivo method for achieving the complete biotinylation of target proteins using a single plasmid co-expressing BirA and its target proteins. Specifically, a biotin-modified protein was produced in Escherichia coli strain BL21(DE3) using a single plasmid containing genes encoding both BirA and a protein fused to BirA's substrate sequence, Avitag. This approach simplifies the production of biotinylated proteins in E. coli and allows the creation of various biotinylated protein types through gene replacement. Furthermore, the biotin modification rate of the obtained target protein could be evaluated using Native-PAGE without performing complicated isolation operations of biotinylated proteins. In Native-PAGE, biotin-modified proteins and unmodified proteins were confirmed as clearly different bands, and it was possible to easily derive the modification rate from the respective band intensities.

Effect of enzymatic modification on the structure and rheological properties of diluted alkali-soluble pectin fraction rich in RG-I

This study focuses on pectin covalently linked in cell walls from two sources, apples and carrots, that was extracted using diluted alkali, and it describes changes in the rheological properties of diluted alkali-soluble pectin (DASP) due to enzymatic treatment. Given DASP’s richness of rhamnogalacturonan I (RG-I), RG-I acetyl esterase (RGAE), rhamnogalacturonan endolyase (RGL), and arabinofuranosidase (ABF) were employed in various combinations for targeted degradation of RG-I pectin chains. Enzymatic degradations were followed by structural studies of pectin molecules using atomic force microscopy (AFM) as well as measurements of rheological and spectral properties. AFM imaging revealed a significant increase in the length of branched molecules after incubation with ABF, suggesting that arabinose side chains limit RG-I aggregation. Structural modifications were confirmed by changes in the intensity of bands in the pectin fingerprint and anomeric region on Fourier transform infrared spectra. ABF treatment led to a decrease in the stability of pectic gels, while the simultaneous use of ABF, RGAE, and RGL enzymes did not increase the degree of aggregation compared to the control sample. These findings suggest that the association of pectin chains within the DASP fraction may rely significantly on intermolecular interactions. Two mechanisms are proposed, which involve side chains as short-range attachment points or an extended linear homogalacturonan conformation favoring inter-chain interactions over self-association.

Luminescence of CsLa1-хCeхSiS4 thiosilicate solid solution: From STE to Ce3+ emission

A series of single crystals of CsLa1-хСехSiS4 monophasic solid solution (x = 0–1) has been obtained for the first time by high-temperature flux synthesis. Methods of XRD and chemical analysis, absorption and low-temperature (from T = 5 K) luminescent spectroscopy were used. The results of the band scheme calculating using density functional theory correlate with spectroscopy data. One non-elementary d → f emission band of Ce3+ ions is observed in the region of 520 nm in the luminescence spectra at room temperature at any value of the x parameter. The luminescence decay kinetics of Ce3+ ions upon excitation by a pulsed electron beam, X-ray synchrotron radiation or intracenter photoexcitation is characterized by a nanosecond component. As the parameter x increases, the decay time is reduced from 132 ns (x = 0.005) to 0.88 ns (x = 1). The luminescence decay kinetics upon photoexcitation at x = 0.005–0.12 is characterized by monoexponential decay with τ = 31.4 ± 0.2 ns. Concentration quenching of the Ce3+ ion photoluminescence is not observed up to the value of the parameter x = 0.12; it only appears at x = 1. The anomalously short decay time of the Ce3+ ions luminescence in CsCeSiS4 upon both X-ray excitation and photoexcitation is associated with concentration quenching.At temperature 5 K, new intense bands at 408 and 688 nm in addition to the Ce3+ emission band observed in the photoluminescence spectra of nominally pure CsLaSiS4 or at the lowest parameter value x = 0.005. These bands correspond to the luminescence of self-trapped excitons (STE) and defects. With increasing x parameter, the STE emission band is reabsorbed by the absorption of Ce3+ ions and is quenched due to the resonance energy transfer STE → Ce3+ center. Thermoluminescence glow curves of CsLa1-xCexSiS4 irradiated with X-ray at T = 90 K are characterized by several low temperature intense peaks, which indicates a high concentration of charge carrier traps.

Luminescence Dynamics of BaAl2O4:Eu2+ Phosphor.

We studied steady-state and time-resolved photoluminescence of Eu doped BaAl2O4 phosphor. The undoped BaAl2O4 sample shows a dominant blue emission band at ~ 428nm and two secondary maxima at ~ 405 and 456nm due to F-centre and aggregate defects such as F2 -centre. The samples after doping of Eu at 1-5% show additional emission bands at ~ 485 and 518 nm due to Eu2+ centre and a red emission band at ~ 657nm is attributed to Eu3+ centre. The sample doped with 2% of Eu shows anomalous emission having the dominant peak at ~ 494nm. The average luminescence lifetime of the emission band at ~ 428nm in the undoped sample was estimated to be (3.29 ± 0.91) ns. The average luminescence lifetime of this emission band after doping of Eu was found to increase by 102 orders of magnitude. The intensity of the 428nm blue emission band was found to quench after doping of Eu beyond 3%. The concentration quenching effect was attributed to dipole-quadrupole interaction. Further, a non-radiative fluorescence energy transfer mechanism from an extrinsic Eu2+ centre to an intrinsic F-centre is proposed to describe the luminescence dynamics of the samples.

Enhanced UVB 311 emission in Sb2O3–SiO2 glasses doped with Gd2O3 and tailored Pb3O4 content for potential phototherapy

This study investigates the enhancement of the sharp 311 nm UVB emission from Gd3+ ions in antimony silicate glass system doped with a fixed concentration of Gd2O3 (1.0 mol%) and varying amounts of red lead (40–10 mol%). The emission spectra revealed an intense sharp UVB band at 311 nm, originating from the 6P7/2 → 8S7/2 transition of gadolinium ions upon excitation at 273 nm. Interestingly, the intensity of this band nearly doubles as the Pb3O4 content decreases from 40 to 10 mol%. Such improvement was attributed to the decomposition of Pb3O4 into various phases that include α-PbO2, β-PbO2 polymorphs, and PbO. Out of these decomposition products, PbO acted as modifiers, reduced phonon losses and consequently enhanced the emission intensity. The decrease in Pb3O4 content is also hypothesized to promote de-clustering of Gd3+ ions within the glass matrix. This de-clustering reduced cross-relaxation losses, further contributed to the observed emission enhancement. In summary, this study demonstrated a successful strategy for boosting the UVB emission from Gd3+ ions in antimony silicate glasses by manipulating the content of Pb3O4.

MODIFICATION OF PINDIGA BENTONITE CLAY WITH DIDODECYLDIMETHYLAMMONIUM BROMIDES SURFACTANT FOR FOUNDRY USE

Bentonite clay from Pindiga, Gombe State, Nigeria, was surface modified with didodecyldimethylammonium bromides (DDAB) by the Base Exchange (cation exchange capacity) method for its possible use in foundries. The surfaces of the unmodified and modified Pindiga bentonite clay were characterised by means of spectroscopic, diffraction, and scanning methods (X-Ray fluorescence spectrometry (XRF)Fourier-transformed infrared spectroscopy (FTIR), X-ray diffraction (XRD), and scanning electron microscopy (SEM).XRF showed that unmodified sample was rich of montmorillonite (MMt) with ca +2 as exchangeable cation, FTIR analysis revealed that unmodified water absorption causes stretching and bending vibrations for hydroxyl groups, with the most intense bands in the low-frequency region, while DDAB organo-bentonites showed slight changes in wave bands, with a strong methyl band and a weak methyl band indicative of long-chain linear aliphatic structure. The basal spacing of the unmodified bentonite sample was 1.52 nm, and after modification with DDAB, it improved to 2.1 nm as determined from the X-ray diffraction (XRD) analysis. Results of the SEM images revealed rougher surfaces with uniformly dispersed small particles of grain-like structures characteristic of the unmodified, while theorgano-modified bentoniterevealed clusters of different sizes and shapes, larger Fe 3+ ions replacing smaller Al 3+ ions at octahedral sites, discontinuous layers enveloping large grains, polygonal growth foams with octahedral and tetrahedral shapes, mixed layers of calcium bentonite, and particle sizes less than 200nm. The study concluded that modified bentonite with didodecyldimethylammonium bromides gives good microstructure, increases rigidity, and improve thermal stability, to the organoclays. Therefore, it is recommended that DDAB bentonite be used as binder for foundries.

Chirality Sensing of Cryptochiral Guests with Prism[n]arenes.

Optical chirality sensing has gained significant attention in recent years. Within this field, the quest for stereodynamic chiroptical probes capable of detecting cryptochiral guests presents a formidable challenge. Macrocycles exhibiting planar chirality have emerged as promising candidates for amplifying the chirality of cryptochiral guests. In this study, we demonstrate that the formation of host-guest complexes between cryptochiral molecules and planar chiral prismarenes triggers electronic circular dichroism (ECD) signals via host-guest complexation-induced chirality amplification. The absolute configuration of the most stable chiral macrocyclic host-guest complex has been established by resorting to both exciton model and DFT computations. Furthermore, we demonstrated that this supramolecular chirality sensing system can be employed to determine the enantiomeric composition of scalemic mixtures by measuring the ECD bands intensity. The information described here opens the way for the use of prismarenes as stereodynamic probes for sensing of cryptochiral guests.

Raman spectroscopy‐based molecularly imprinted polymer sensor for sensitive detection of lysophosphatidic acid in serum

AbstractLysophosphatidic acid (LPA) has significant potential as a biomarker for diagnosis of ovarian cancer in female patients because it is present throughout all phases of the illness. Regrettably, there is not a clinically useful test for this biomarker yet. In this work, an ultra‐detection of LPA molecule by Raman spectroscopy‐based molecularly imprinted polymer (MIP) in serum samples is reported. β‐CD was synthesized to facilitate the collection of Raman spectroscopy and combined with MIP to denote it as β‐CD‐MIP for the adsorption of LPA with high sensitivity and selectivity. Linear regression model in serum samples shows peaks at 1100 and 1116 cm−1, respectively. A satisfactory linear fit was seen with LPA concentration log I = 3.7 log C + 1255.74 and with spiked serum log I = 4.6 log C + 327.22, respectively. The literature survey suggests the threshold LPA level in healthy person is 1.50 μM/L whereas the LPA level rises in patients with benign ovarian cancer tumor as 7.90 μM/L. This LPA level rises to 16.99 μM/L in ovarian cancer patients. On the basis of the band intensity, the limit of detection of the β‐CD‐MIP sensor for LPA was recorded as 0.003 μM/L. The binding constant of β‐CD‐MIP for LPA in serum was 0.031–0.003 μM/L, respectively. β‐CD‐MIP gel was optimized, characterized, and successfully applied on spiked serum samples. The findings of this research show that this substrate has a lot of potential for ultradetection of trace amounts of LPA molecule from the serum samples. The MIP sensor is robust, recyclable, efficient, and selective.

Changes in characteristics of spermatogonial stem cells in response to heat stress in stallions

Spermatogonial stem cells (SSCs) are essential for the maintenance of male fertility and survival of species. Environmental conditions, notably heat stress, have been identified as important causes of male infertility and have a negative impact on SSCs. Animals with cryptorchid testes (CT) are optimal models for the study of long-term heat stress-related changes in germ cells. The effect of heat stress on germ cells differs depending on the spermatogenesis stage. Thus, verifying whether the specific phase of spermatogenesis is dependent or independent of heat stress in stallions is important. We evaluated the heat stress-related response of SSCs by comparing the relative abundance of mRNA transcripts and expression patterns of the undifferentiated embryonic cell transcription factor 1 (UTF-1) and deleted in azoospermia-like (DAZL) in the seminiferous tubules of CT and normal testes (NT) of stallions using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), immunofluorescence, and western blotting. We also analyzed the relative abundance of mRNA of different proliferative markers, including minichromosome maintenance 2 (MCM2), marker of proliferation Ki-67 (MKI-67), and proliferating cell nuclear antigen (PCNA). Testicular tissues from four Thoroughbred unilateral cryptorchid postpubertal stallions were used in this study during the breeding season. The relative abundance of the mRNA transcripts of UTF-1 and MCM2 was significantly upregulated in the CT group than that of those in the NT group. In contrast, the relative abundance of the mRNA transcripts of DAZL was significantly downregulated in the CT group than that of those in the NT group. Western blot quantification showed that the relative intensity of UTF-1 protein bands was significantly higher, while that of DAZL protein bands was significantly lower in the CT group than in the NT group. Immunofluorescence studies showed that the number of germ cells immunostained with UTF-1 was significantly higher while immunostained with DAZL was significantly lower in the CT group than that in the NT group. The higher expression level of UTF-1 in the CT group shows that undifferentiated SSCs are not affected by long-term exposure to heat stress. These results also indicate that germ cells after differentiation phase are directly affected by heat-stress conditions, such as cryptorchidism, in stallions.

Observation of exceptionally strong near-bottom flows over the Atlantis II Seamounts in the northwest Atlantic

Knowledge of near-bottom ocean current velocities and especially their extreme values is necessary to understand geomorphology of the seafloor and composition of benthic biological communities and quantify mechanical energy dissipation by bottom drag. Direct measurements of near-bottom currents in deep ocean remain scarce because of logistical challenges. Here, we report the results of flow velocity and pressure fluctuation measurements at three sites with depths of 2573–4443 m in the area where the Gulf Stream interacts with the New England Seamounts. Repeated episodes of unexpectedly strong near-bottom currents were observed, with the current speed at 4443 m of more than 0.40 m/s. At 2573 m, current speeds exceeded 0.20 m/s approximately 5% of the time throughout the entire eight-week measurement period. The maximum flow speeds of over 1.10 m/s recorded at this site significantly surpass the fastest previously reported directly measured current speeds at comparable or larger depths. A strong correlation is found between the noise intensity in the infrasonic band and the measured current speed. The noise intensity and the characteristic frequency increase with the increasing current speed. Machine-learning tools are employed to infer current speeds from flow-noise measurements at the site not equipped with a current meter.

Associations of accelerometer-estimated free-living daily activity impact intensities with 10-year probability of osteoporotic fractures in adults

PurposeAccelerometers are used to objectively measure physical activity; however, the relationship between accelerometer-based activity parameters and bone health is not well understood. This study examines the association between accelerometer-estimated daily activity impact intensities and future risk estimates of major osteoporotic fractures in a large population-based cohort. MethodsParticipants were 3165 adults 46 years of age from the Northern Finland Birth Cohort 1966 who agreed to wear a hip-worn accelerometer during all waking hours for 14 consecutive days. Raw accelerometer data were converted to resultant acceleration. Impact magnitude peaks were extracted and divided into 32 intensity bands, and the osteogenic index (OI) was calculated to assess the osteogenic effectiveness of various activities. Additionally, the impact peaks were categorized into three separate impact intensity categories (low, medium, and high). The 10-year probabilities of hip and all major osteoporotic fractures were estimated with FRAX-tool using clinical and questionnaire data in combination with body mass index collected at the age of 46 years. The associations of daily activity impact intensities with 10-year fracture probabilities were examined using three statistical approaches: multiple linear regression, partial correlation, and partial least squares (PLS) regression. ResultsOn average, participants’ various levels of impact were 8331 (SD = 3478) low; 2032 (1248) medium; and 1295 (1468) high impacts per day. All three statistical approaches found a significant positive association between the daily number of low-intensity impacts and 10-year probability of hip and all major osteoporotic fractures. In contrast, increased number of moderate to very high daily activity impacts was associated with a lower probability of future osteoporotic fractures. A higher OI was also associated with a lower probability of future major osteoporotic fractures. ConclusionLow-intensity impacts might not be sufficient for reducing fracture risk in middle-aged adults, while high-intensity impacts could be beneficial for preventing major osteoporotic fractures.

Yb3+ concentration influence on NIR and upconversion emission and temperature sensing properties of Er3+/Yb3+ co-doped Ta2O5 particles

We synthesized Er3+ and Yb3+ co-doped tantalum oxide (Ta2O5) spherical sub-micrometric particles by a facile method involving formation of tantalum glycolates after pouring in acetone. Then, we investigated how the annealing temperature and Yb3+ concentration (0–30 mol %) affect their structural and luminescence properties. Particle sizes ranged from 146 to 258 nm for the samples annealed at 800 °C. XRD analysis revealed the orthorhombic L-Ta2O5 crystalline structure. The different symmetry sites that the rare earth ions can occupy within the Ta2O5 structure led to intense and broad emission bands centered around 1530 nm. Up-conversion measurements and the calculated number of photons for intense green and red emissions obtained with 980 and 1550 nm excitations helped us to elucidate the mechanisms involved at both excitation wavelengths. We investigated how the 0.5 mol % Er3+/1.5 mol % Yb3+ co-doped Ta2O5 sample performs as a primary thermometer by using the Boltzmann distribution law to predict the absolute temperature from the ratio between the Er3+ 2H11/2 → 4I15/2 and 4S3/2 → 4I15/2 transitions. We obtained a maximum relative thermal sensitivity of 0.97 ± 0.04 % K−1 and a minimum δT of 2.41 K. The calculated repeatability was above 95%. These results show that promising nanothermometer based on Ta2O5 with controlled size and morphology can be designed.

Simple and sensitive galactose monitoring based on capillary SERS sensor.

Galactosemia, a severe genetic metabolic disorder, results from the absence of galactose-degrading enzymes, leading to harmful galactose accumulation. In this study, we introduce a novel capillary-based surface-enhanced Raman spectroscopy (SERS) sensor for convenient and sensitive galactose detection. The developed sensor enhances SERS signals by introducing gold nanoparticles (Au NPs) onto the surface of silver nanoshells (Ag NSs) within a capillary, creating Ag NSs with Au NPs as satellites. Utilizing 4-mercaptophenylboronic acid (4-MPBA) as a Raman reporter molecule, the detection method relies on the conversion of 4-MPBA to 4-mercaptophenol (4-MPhOH) driven by hydrogen peroxide (H2O2) generated during galactose oxidation by galactose oxidase (GOx). A new SERS signal was observed, which was generated by H2O2 produced when galactose and GOx reacted. Our strategy yielded a quantitative change in the SERS signal, specifically in the band intensity ratio of 998 to 1076 cm-1 (I998/I1076) as the galactose concentration increased. Our capillary-based SERS biosensor provides a promising platform for early galactosemia diagnosis.

The pH effects on thermal amyloid fibrillation kinetics of hen egg‐white lysozyme using new normalization factor for Raman spectroscopy

The <i>p</i>H effects on thermal amyloid fibrillation kinetics of hen egg‐white lysozyme using new normalization factor for Raman spectroscopy

The interactions of pectin with TiO2 nanoparticles measured by FT-IR are confirmed in a model of the gastrointestinal tract

The presence of nanoparticle fractions (<100 nm, NPs) in the food additive TiO2 (E171) rises concerns about its potential harmful impact on human health. The knowledge about the interaction of TiO2 NPs with food components is limited to proteins or polyphenols. The present paper is the first to report on interactions between TiO2 NPs and high molecular pectins that form gels in boluses and are remain nearly intact during digestion until they reach the colon. Direct interactions were studied using Fourier Transform Infrared Spectroscopy while indirect ones were monitored by measuring the “absorption” of TiO2 using a 0.2 microfiltration membrane, during in vitro digestion in a model of the gastro-intestinal tract. The FT-IR spectra registered for pectin-TiO2 NPs solutions confirmed changes in band intensities at 1020, 1100, 1610, and 1740 cm−1, suggesting interactions taking place mainly via the COO– groups. Furthermore, the I(1020)/I(1100) ratio was decreased (C-O stretching vibrations), suggesting partial blocking of the skeletal vibrations caused by interactions between pectin and TiO2. The modelled in vitro digestions confirmed that the “availability” of Ti was reduced when TiO2 NPs were combined with pectin, as compared to TiO2 NPs “digested” alone.

Optical properties of nanocomposites based on polypropylene modified by nanoparticles of oxide compounds

The optical properties in UV, visible and near IR spectral regions for nanocomposites based on polypropylene modified by nanoparticles of oxide compounds ZrO2, Al2O3, SiO2, MgO, TiO2, ZnO were studied. A shift of the main optical absorption edge to the short-wave region upon modification by dielectric nanoparticles (MgO, ZrO2, and Al2O3) was established. The absorption edge shifts to the long-wave region upon the introduction of semiconductor nanoparticles (ZnO, TiO2) and amorphous SiO2 particles. In all spectra, the absorption bands at 1200, 1400, and 1720 nm characterized by overtone vibrations of CH, CH2, and CH3 molecular groups were recorded. OH groups make a certain contribution to the bands at 1400 and 1720 nm. The dependence of the intensity of these absorption bands on the type and concentration of nanoparticles used in the modification of polypropylene was analyzed. X-ray diffraction analysis of the studied nanocomposites was carried out, the formation of β-PP was registered upon modification by Al2O3 nanoparticles. Photoluminescence spectra of nanocomposites were studied. It was found that the introduction of nanoparticles (except SiO2) does not lead to the formation of new luminescence bands in PP

Heat Treatment and Homogenization Influence the Gastric Digestion of Bovine Milk Protein in Growing Pigs as an Adult Human Model

BackgroundBovine milk processing influences the structure of the curd formed during gastric digestion, which may alter gastric protein hydrolysis and impact amino acid (AA) release into the small intestine. ObjectivesThis study aimed to determine the influence of heat treatment and homogenization on the gastric protein digestion and AA emptying of bovine milk. MethodsNine-wk-old pigs (n = 144) consumed either raw, pasteurized nonhomogenized (PNH), pasteurized homogenized (PH), or ultrahigh- temperature homogenized (UHT) bovine milk for 10 d. On day 11, fasted pigs received the milk treatment (500 mL) before gastric contents were collected at 0, 20, 60, 120, 180, and 300 min postprandially. The apparent degree of gastric protein hydrolysis (based on the release of free amino groups), apparent gastric disappearance of individual proteins [based on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel band intensity], and the gastric emptying of digested protein and AA were determined. ResultsDuring the first 60 min, the rate of apparent gastric protein hydrolysis was fastest in pigs fed UHT milk (0.29%/min compared with on average 0.07%/min in pigs fed raw, PNH, and PH milk). Differences in the apparent degree of gastric protein hydrolysis and emptying were reflected in the rate of digested protein entering the small intestine. The AA gastric emptying half-time was generally shorter in pigs fed PH and UHT milk than pigs fed raw and PNH milk. For example, the gastric release of total essential AAs was >2-fold faster (P < 0.01) in pigs fed PH or UHT milk than that of pigs fed raw or PNH milk (i.e., homogenized compared with nonhomogenized milk). ConclusionsHeat treatment and homogenization increased the apparent gastric degree of protein hydrolysis and the release of digested protein into the small intestine. However, the rate of AAs entering the small intestine was mainly increased by homogenization.

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.

Using salted egg white in steamed bread: Impact on functional and structural characteristics

Steamed bread has long been an important part of Chinese cuisine. This study investigated the effects of salted egg white (SEW) (5, 10, 15, and 20% w/w) on the quality of steamed breads. Findings revealed that SEW notably enhanced the bread's volume and texture, with a 20% inclusion significantly boosting water retention and rheological properties, albeit reducing bread's lightness. In addition, the H-bond absorption band intensity in the Fourier transform infrared spectroscopy (FTIR) analysis showed increased peak intensities with higher SEW levels, indicative of protein structure alterations. X-ray diffraction confirmed the presence of an amylose–lipid complex. Scanning electron microscope (SEM) and Confocal laser scanning microscope (CLSM) imaging depicted a smooth, consistent protein network with SEW addition. Consumer sensory evaluation responded favourably to the SEW15 steamed bread, suggesting its potential for food industry application. Overall, the study considers SEW an effective ingredient for improving steamed bread quality.

Using Biotinylated Iron-Responsive Element to Analyze the Activity of Iron Regulatory Proteins.

Iron regulatory proteins (IRP1 and IRP2) are the master regulators of mammalian iron homeostasis. They bind to the iron-responsive elements (IREs) of the transcripts of iron-related genes to regulate their expression, thereby maintaining cellular iron availability. The primary method to measure the IRE-binding activity of IRPs is the electrophoresis mobility shift assay (EMSA). This method is particularly useful for evaluating IRP1 activity, since IRP1 is a bifunctional enzyme and its protein levels remain similar during conversion between the IRE-binding protein and cytosolic aconitase forms. Here, we exploited a method of using a biotinylated-IRE probe to separate IRE-binding IRPs followed by immunoblotting to analyze the IRE-binding activity. This method allows for the successful measurement of IRP activity in cultured cells and mouse tissues under various iron conditions. By separating IRE-binding IRPs from the rest of the lysates, this method increases the specificity of IRP antibodies and verifies whether a band represents an IRP, thereby revealing some previously unrecognized information about IRPs. With this method, we showed that the S711-phosphorylated IRP1 was found only in the IRE-binding form in PMA-treated Hep3B cells. Second, we found a truncated IRE-binding IRP2 isoform that is generated by proteolytic cleavage on sites in the 73aa insert region of the IRP2 protein. Third, we found that higher levels of SDS, compared to 1-2% SDS in regular loading buffer, could dramatically increase the band intensity of IRPs in immunoblots, especially in HL-60 cells. Fourth, we found that the addition of SDS or LDS to cell lysates activated protein degradation at 37 °C or room temperature, especially in HL-60 cell lysates. As this method is more practical, sensitive, and cost-effective, we believe that its application will enhance future research on iron regulation and metabolism.