Comparison of the N-acetylhexosaminidase components in the ram testis and epididymis by isoelectric focusing.
Ontology highlight
ABSTRACT:
SUBMITTER: Winchester BG
PROVIDER: S-EPMC1177281 | biostudies-other | 1971 Oct
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Three and four N-acetylhexosaminidase components, from ram testis and epididymis respectively, have been separated by ion-exchange chromatography on DEAE-cellulose. Although they all have the same molecular weight (approx. 140000) and very similar catalytic properties towards the synthetic substrates, 4-methylumbelliferyl N-acetyl-beta-glucosaminide and N-acetyl-beta-galactosaminide, isoelectric focusing of the individual components showed that each had a distinct pI value. Isoelectric focusing has also been used to demonstrate the occurrence of multiple forms in ejaculated ram semen.
| S-EPMC1177738 | biostudies-other
Project description:Mitochondria are highly heterogeneous organelles that likely have unique isoelectric points (pI), which are related to their surface compositions and could be exploited in their purification and isolation. Previous methods to determine pI of mitochondria report an average pI. This article is the first report of the determination of the isoelectric points of individual mitochondria by capillary isoelectric focusing (cIEF). In this method, mitochondria labeled with the mitochondrial-specific probe 10-N-nonyl acridine orange (NAO) are injected into a fused-silica capillary in a solution of carrier ampholytes at physiological pH and osmolarity, where they are focused then chemically mobilized and detected by laser-induced fluorescence (LIF). Fluorescein-derived pI markers are used as internal standards to assign a pI value to each individually detected mitochondrial event, and a mitochondrial pI distribution is determined. This method provides reproducible distributions of individual mitochondrial pI, accurate determination of the pI of individual mitochondria by the use of internal standards, and resolution of 0.03 pH units between individual mitochondria. This method could also be applied to investigate or design separations of organelle subtypes (e.g., subsarcolemmal and interfibrillar skeletal muscle mitochondria) and to determine the pIs of other biological or nonbiological particles.
| S-EPMC3021621 | biostudies-literature
Project description:This work presents the first implementation of cascaded stages for a microfabricated free-flow isoelectric focusing (FF-IEF) device. Both analytical and computational models for IEF suggest device performance will be improved by utilizing multiple stages to reduce device residence time. These models are shown to be applicable by using focusing of small IEF markers as a demonstration. We also show focusing of fluorescently tagged proteins under different channel geometries, with the most efficient focusing occurring in the cascaded design, as predicted by theory. An additional aim of this work is to demonstrate the compatibility of cascaded FF-IEF with common bioanalytical tools. As an example, outlet fractions from cascaded FF-IEF were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Processing of whole cell lysate followed by immunoblotting for cell signaling markers demonstrates the reduction of albumin from samples, as well as the enrichment of apoptotic markers.
| S-EPMC2560584 | biostudies-literature
Project description:We describe a novel isoelectric point photoswitching phenomenon in both wild-type Aequorea victoria (av) GFP and the amino acid 222 E-to-G mutant Aequorea coerulescens (ac) GFP. A combination of time-resolved microfluidic isoelectric focusing (IEF) and in situ antibody blotting IEF was employed to monitor dark (nonfluorescent) and bright (fluorescent) GFP populations. Through IEF, each population was observed to exhibit distinct isoelectric points (pI) and, thus, distinct formal electrostatic charges. Experimentally observed interconversion between the dark, higher pI and bright, lower pI GFP populations is tightly controlled by differential UV and blue light exposure. The stoichiometry and kinetics of charge transfer tied to this reversible photobleaching process are deduced. In concert with a reaction-transport model of bistable reversible charge and fluorescence photoswitching, the on-chip measurements of population interconversion rates suggest the potential for both rheostatic and discrete switch-like modulation of the electrostatic charge of GFPs depending on the illumination profile. We estimate that 3-4 formal charges distinguish the bright and dark populations of avGFP, as compared to one charge for those of acGFP. Given the proposed role of E222 as a bridge between internal and exit hydrogen-bond clusters within the GFP ?-barrel, the difference in charge switching magnitude between the two mutants provides intriguing evidence for the proton wire hypothesis of proton transport within the GFP structure, and of proton exchange with the bulk solvent. Our facile dynamic and probed IEF assays should find widespread use in analytical screening and quantitative kinetic analysis of photoswitching and other charge switching processes in response to stimuli including light, temperature, or binding/cleavage events.
| S-EPMC3488114 | biostudies-literature
Project description:Isoelectric focusing (IEF) is a powerful separation method, useful for resolving subtle changes in the isoelectric point of unlabeled proteins. While microfluidic IEF has reduced the separation times from hours in traditional benchtop IEF to minutes, the enclosed devices hinder post-separation access to the sample for downstream analysis. The two-layer open IEF device presented here comprises a photopatterned hydrogel lid layer containing the chemistries required for IEF and a thin polyacrylamide bottom layer in which the analytes are separated. The open IEF device produces comparable minimum resolvable difference in isoelectric point and gradient stability to enclosed microfluidic devices while providing post-separation sample access by simple removal of the lid layer. Further, using simulations, we determine that the material properties and the length of the separation lanes are the primary factors that affect the electric field magnitude in the separation region. Finally, we demonstrate self-indexed photomasks for alignment-free fabrication of multi-domain hydrogels. We leverage this approach to generate arrayed pH gradients with a total of 80 concurrent separation lanes, which to our knowledge is the first demonstration of multiple IEF separations in series addressed by a single pair of electrodes.
| S-EPMC6106862 | biostudies-literature
Project description:Immunoprobed isoelectric focusing (IEF) resolves proteins based on differences in isoelectric point (pI) and then identifies protein targets through immunoprobing of IEF-separated proteins that have been immobilized onto a gel scaffold. During the IEF stage, the gel functions as an anti-convective medium and not as a molecular sieving matrix. During the immunoprobing stage, the gel acts as an immobilization scaffold for IEF-focused proteins via photoactive moieties. Here, we characterized the effect of gel pore size on IEF separation and in-gel immunoassay performance. We modulated polyacrylamide (PA) gel pore size via lateral chain aggregation initiated by PEG monomers. During IEF, the 2% PEG highly porous PA gel formulation offered higher resolution (minimum pI difference ∼0.07 ± 0.02) than unmodified 6%T, 3.3%C (benchmark) and 6%T, 8%C (negative control) PA gels. The highly porous gels supported a pH gradient with slope and linearity comparable to benchmark gels. The partition coefficient for antibodies into the highly porous gels (K = 0.35 ± 0.02) was greater than the benchmark (3×) and negative control (1.75×) gels. The highly porous gels also had lower immunoassay background signal than the benchmark (2×) and negative control (3×) gels. Taken together, lateral aggregation creates PA gels that are suitable for both IEF and subsequent in-gel immunoprobing by mitigating immunoprobe exclusion from the gels while facilitating removal of unbound immunoprobe.
| S-EPMC7861876 | biostudies-literature
Project description:A previously undescribed isoelectric focusing technology allows cell signaling to be quantitatively assessed in <25 cells. High-resolution capillary isoelectric focusing allows isoforms and individual phosphorylation forms to be resolved, often to baseline, in a 400-nl capillary. Key to the method is photochemical capture of the resolved protein forms. Once immobilized, the proteins can be probed with specific antibodies flowed through the capillary. Antibodies bound to their targets are detected by chemiluminescence. Because chemiluminescent substrates are flowed through the capillary during detection, localized substrate depletion is overcome, giving excellent linearity of response across several orders of magnitude. By analyzing pan-specific antibody signals from individual resolved forms of a protein, each of these can be quantified, without the problems associated with using multiple antibodies with different binding avidities to detect individual protein forms.
| S-EPMC1618307 | biostudies-literature
Project description:Pressure-driven remobilization without an applied electric field is shown to be possible with capillary isoelectric focusing using packed capillaries. The capillary dimensions are 100 ?m i.d. and 2 cm in length, and the packing is made of 0.9 ?m nonporous silica particles that are chemically modified with a brush layer of polyacrylamide. Both reversible and irreversible adsorption are shown to be negligible. The packed capillaries eliminate the problem of unwanted hydrodynamic flow between reservoirs. Three proteins are focused: trypsin inhibitor, carbonic anhydrase II, and myoglobin. The time required for focusing in the packed capillaries is increased by only a factor of 2 compared to the open capillary, giving complete focusing in less than 15 min at 200 V/cm. The packed capillaries allow the use of higher electric fields, with resolution continually increasing up to at least 1500 V/cm. The packing obstructs diffusional broadening after the field is turned off: for trypsin inhibitor, D = 6.1(±0.3) × 10(-8) cm(2)/s for the packed capillary vs D = 28.8(±0.3) × 10(-8) cm(2)/s for the open capillary. The broadening contributed by the packing during remobilization is from eddy diffusion, and it is described by its plate height, H, which is the variance per unit length: H = ?(2)/L = 0.64 ?m. This limits the resolution to 0.1 pH units for the 2 cm capillary having a pH range of 3-10, giving a theoretical peak capacity of 47.
| S-EPMC3042039 | biostudies-literature
Project description:The human testis and epididymis play critical roles in male fertility, including the spermatogenesis process, sperm storage, and maturation. However, the unique functions of the two organs had not been systematically studied. Herein, we provide a systematic and comprehensive multi-omics study between testis and epididymis. RNA-Seq profiling detected and quantified 19,653 in the testis and 18,407 in the epididymis. Proteomic profiling resulted in the identification of a total of 11,024 and 10,386 proteins in the testis and epididymis, respectively, including 110 proteins that previously have been classified as MPs (missing proteins). Furthermore, Five MPs expressed in testis were validated by the MRM method. Subsequently, multi-omcis between testis and epididymis were performed, including biological functions and pathways of DEGs (Differentially Expressed Genes) in each group, revealing that those differences were related to spermatogenesis, male gamete generation, as well as reproduction. In conclusion, this study can help us find the expression regularity of missing protein and help related scientists understand the physiological functions of testis and epididymis more deeply.
| S-EPMC8199593 | biostudies-literature
Project description:1. Isoelectric focusing of human liver alkaline phosphatase in a sucrose density gradient with LKB Ampholine as carrier ampholytes is described. 2. Problems due to the chelating properties of the ampholytes and the pH gradient were examined. 3. A reactivation procedure to counter these effects was devised that can probably be used for other alkaline phosphatases. 4. The isoelectric point of human liver alkaline phosphatase was found to be pH3.9.
| S-EPMC1179117 | biostudies-other