An apparent molecular weight (MW) of a protein can be determined from the migration distance of a protein complexed with a strong cationic detergent sodium dodecyl sulfate (SDS) separated on sodium ...dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This method was established around 1969 and has been utilized substantially even today because of its simplicity. During the following half a century, although it has been reported that many proteins show some deviation in MW when determined on SDS-PAGE especially when their peptide chains are posttranslationally modified, this versatile method is still being used very often in current biochemical works. In this protocol, a simple method to estimate MW by running SDS-PAGE of standard proteins is explained by an example in which proteins extracted from mouse retina were analyzed by two-dimensional isoelectric focusing (2-D IEF) SDS-PAGE followed by protein identification by peptide mass fingerprinting.
Mucuna pruriens (L.) DC var. pruriens is a well-known legume of Fabaceae family. The plant is the natural source of l-DOPA, precursor molecule of dopamine neurotransmitter used in the treatment of ...Parkinson´s disease. In M. pruriens, the Catecholamine (CA) biosynthesis is mediated by Polyphenol oxidase (PPO) as validated in our previous reports. In the present investigation, we identified three PPO isoforms and were quantitatively analyzed by quantitative real-time PCR to understand the expression of PPO´s constitutively in the different tissues of M. pruriens. Consequently, the highly expressed PPO isoform was cloned, and the recombinant protein purified followed by functional characterization. The Kinetic analysis using Lineweaver-Burk curves showed greater binding affinity of enzyme towards its specific substrate. This study provides new insight into plant derived recombinant PPO enzyme, which is involved in CA biosynthetic pathway in this taxon. The Metabolic pathway engineering of PPO step would result in modulation of l-DOPA synthesis in this plant, in future.
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•Identified and confirmed the existence of PPO as isoforms in M. pruriens.•Quantitative expression of the highest expressed isoform using qRT-PCR.•Functional characterization of recombinantly expressed purified PPO.
Sodium dodecyl sulfate-sedimentation volume is an important index to evaluate the gluten strength of common wheat and is closely related to baking quality. In this study, a total of 15 quantitative ...trait locus (QTL) for sodium dodecyl sulfate (SDS)-sedimentation volume (SSV) were identified by using a high-density genetic map including 2,474 single-nucleotide polymorphism (SNP) markers, which was constructed with a doubled haploid (DH) population derived from the cross between Non-gda3753 (ND3753) and Liangxing99 (LX99). Importantly, four environmentally stable QTLs were detected on chromosomes 1A, 2D, and 5D, respectively. Among them, the one with the largest effect was identified on chromosome 1A (designated as
) explaining up to 39.67% of the phenotypic variance. Subsequently,
was dissected into two QTLs named as
and
by saturating the genetic linkage map of the chromosome 1A. Interestedly, favorable alleles of these two loci were from different parents. Due to the favorable allele of
was from the high-value parents ND3753 and revealed higher genetic effect, which explained 25.07% of the phenotypic variation, mapping of this locus was conducted by using BC
F
and BC
F
populations. By comparing the CS reference sequence, the physical interval of
was delimited into 14.9 Mb, with 89 putative high-confidence annotated genes. SSVs of different recombinants between
and
detected from DH and BC
F
populations showed that these two loci had an obvious additive effect, of which the combination of two favorable loci had the high SSV, whereas recombinants with unfavorable loci had the lowest. These results provide further insight into the genetic basis of SSV and
will be an ideal target for positional cloning and wheat breeding programs.
Understanding near infrared light propagation in tissue is vital for designing next generation optical brain imaging devices. Monte Carlo (MC) simulations provide a controlled mechanism to ...characterize and evaluate contributions of diverse near infrared spectroscopy (NIRS) sensor configurations and parameters. In this study, we developed a multilayer adult digital head model under both healthy and clinical settings and assessed light‐tissue interaction through MC simulations in terms of partial differential pathlength, mean total optical pathlength, diffuse reflectance, detector light intensity and spatial sensitivity profile of optical measurements. The model incorporated four layers: scalp, skull, cerebrospinal‐fluid and cerebral cortex with and without a customizable lesion for modeling hematoma of different sizes and depths. The effect of source‐detector separation (SDS) on optical measurements' sensitivity to brain tissue was investigated. Results from 1330 separate simulations (4 lesion volumes × 4 lesion depths for clinical +3 healthy settings) × 7 SDS × 10 simulation = 1330) each with 100 million photons indicated that selection of SDS is critical to acquire optimal measurements from the brain and recommended SDS to be 25 to 35 mm depending on the wavelengths to obtain optical monitoring of the adult brain function. The findings here can guide the design of future NIRS probes for functional neuroimaging and clinical diagnostic systems.
Near infrared spectroscopy (NIRS) neuroimaging sensors use light source and detector pairs placed over scalp to measure underlying neurophysiology. Source detector separation (SDS) is a critical design parameter and should aim a balanced trade‐off for detected light intensity and sensitivity to brain tissue. In this study, we used multilayer digital head models to systematically evaluate the role of SDS on NIRS spatial sensitivity profiles within both healthy and clinical conditions.
Solvent retention capacity (SRC) is a solvation test for wheat flour in which functional contributions of different polymeric components are predicted based on their swelling behavior with different ...diagnostic solvents. To assess the functionality of individual gluten components, four solvents have been proposed: 1) 55% aqueous ethanol for gliadins; 2) 0.75% sodium dodecyl sulfate (SDS) for glutenin macropolymer; 3) 0.006% sodium metabisulfite (MBS) for gluten strength; and 4) 0.75% SDS + 0.006% MBS for glutenin macropolymer without disulfide network. This research aimed to study the gluten functionality of commercial wheat flour samples by the four proposed supplemental diagnostic solvents. Commercial wheat samples were purchased in 2020 at a grocery store in Fargo, North Dakota. Four different brands from three flour types (bread flour, all-purpose flour, and cake flour) were analyzed. These flour types represent wheat flour from three different U.S. wheat classes, hard spring wheat, hard winter wheat, and soft wheat. SRC was measured according to the AACC-approved method 56–11.02 with modifications. The supplementary solvents 55% aqueous ethanol, 0.75% SDS, 0.006% MBS, and 0.75% SDS +0.006% MBS were used instead of the traditional SRC solvents. Highly significant (p ≤ 0.001) and positive correlations between SDS SRC, MBS SRC, and SDS + MBS SRC solvents and the analyzed rheological and baking quality traits, especially water absorption and loaf volume. Additionally, when protein composition was assessed, the three previously mentioned supplementary SRC solvents denoted a significant (p ≤ 0.01) and positive correlation with the glutenin polymers fraction, which is associated with dough functionality. These results highlight the importance of assessing specific flour polymers' functional contributions to predict each flour type's end-use potential.
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•Supplementary solvent retention capacity (S-SRC) solvents target gluten components.•S-SRC solvents showed potential for evaluation of gluten functionality.•S-SRC solvents were highly correlated with water absorption, loaf volume & firmness.•S-SRC 55% ethanol solvent optimization has been proposed for gliadin evaluation.•Glutenin polymers fraction was highly correlated with three S-SRC solvents.
The Two-Way Ranging (TWR) method is commonly used for measuring the distance between two wireless transceiver nodes, especially when clock synchronization between the two nodes is not available. For ...modeling the time-of-flight (TOF) error between two wireless transceiver nodes in TWR, the existing error model, described in the IEEE 802.15.4-2011 standard, is solely based on clock drift. However, it is inadequate for in-depth comparative analysis between different TWR methods. In this paper, we propose a novel TOF Error Estimation Model (TEEM) for TWR methods. Using the proposed model, we evaluate the comparative analysis between different TWR methods. The analytical results were validated with both numerical simulation and experimental results. Moreover, we demonstrate the pitfalls of the symmetric double-sided TWR (SDS-TWR) method, which is the most highlighted TWR method in the literature because of its highly accurate performance on clock-drift error reduction when reply times are symmetric. We argue that alternative double-sided TWR (AltDS-TWR) outperforms SDS-TWR. The argument was verified with both numerical simulation and experimental evaluation results.
The opportunistic pathogen
strain PAO1 is able to use a variety of organic pollutants as growth substrates, including the anionic detergent sodium dodecyl sulfate (SDS) and long-chain alkanes. While ...the enzymes initiating SDS and alkane degradation are well known, the subsequent enzymatic steps for degradation of the derived primary long-chain alcohols have not yet been identified. By evaluating genes specifically induced during growth with SDS, a gene cluster encoding a putative alcohol dehydrogenase (PA0364/LaoA), a probable inner membrane protein (PA0365/LaoB), and a presumable aldehyde dehydrogenase (PA0366/LaoC) was identified and designated the Lao (
ong-chain-
lcohol/
ldehyde-
xidation) system. Growth experiments with deletion mutants with SDS, 1-dodecanol, and alkanes revealed that LaoA and LaoB are involved in the degradation of primary long-chain alcohols. Moreover, detection of 1-dodecanol oxidation in cell extracts by activity staining revealed an interdependency of LaoA and LaoB for efficient 1-dodecanol oxidation. An
analysis yielded no well-characterized homologue proteins for LaoA and LaoB. Furthermore, a gene adjacent to the
gene cluster encodes a putative transcriptional regulator (PA0367/LaoR). A
deletion mutant exhibited constitutive expression of LaoA and LaoB, indicating that LaoR is a repressor for the expression of
Taken together, these results showed that the proteins LaoA and LaoB constitute a novel oxidation system for long-chain alcohols derived from pollutants.
The versatile and highly adaptive bacterium
a is able to colonize a variety of habitats, including anthropogenic environments, where it is often challenged with toxic compounds. Its ability to degrade such compounds and to use them as growth substrates can significantly enhance spreading of this opportunistic pathogen in hygienic settings, such as clinics or water distribution systems. Thus, knowledge about the metabolism of
can contribute to novel approaches for preventing its growth and reducing nosocomial infections. As the Lao system is important for the degradation of two different classes of pollutants, the identification of these novel enzymes can be a useful contribution for developing effective antibacterial strategies.
As an alternative living cell membrane, sodium dodecyl sulfide (SDS) has been investigated at this study, spectroscopic assessment of Finasteride diffusion through the alternate model of the cell ...membrane in two distinct polar solutions, buffer phosphate solution pH 7.4 and Cyclohexane(Non-polar solution), has been studied. Finasteride in buffer phosphate solution showed a definite ?max at 253 nm, which was consistent with the default values. The results also reveal that the non-polar medium has a lower attenuation coefficient and a shorter maximum wavelength than the polar medium. The pH 7.4 finasteride solution demonstrated remarkable stability over time.
Diffusion rate of Finasteride was investigated using Sodium Dodecyl Sulfate (SDS) as an alternative cell membrane model. The chemical potential was determined to be - 6294.76 J mol-1, showing the spontaneity of the compound's diffusion process.
The results indicated that Finasteride may diffuse (at a rate constant of 51.11 min-1) from the aqueous media and other identified components to the inner micelle; the equilibrium constant for diffusion was calculated to be 11.5.
Keywords: Finasteride, Diffusion, Chemical potential, SDS, Alternative model, Cell membrane.
The Western Blot Hnasko, Thomas S; Hnasko, Robert M
Methods in molecular biology (Clifton, N.J.),
2015, Volume:
1318
Journal Article
Western blotting is a technique that involves the separation of proteins by gel electrophoresis, their blotting or transfer to a membrane, and selective immunodetection of an immobilized antigen. ...This is an important and routine method for protein analysis that depends on the specificity of antibody-antigen interaction and is useful for the qualitative or semiquantitative identification of specific proteins and their molecular weight from a complex mixture. This chapter will outline the requisite steps including gel electrophoresis of a protein sample, transfer of protein from a gel to a membrane support, and immunodetection of a target antigen.
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