Isoelectric focusing technology quantifies protein signaling in 25 cells.
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ABSTRACT:
SUBMITTER: O'Neill RA
PROVIDER: S-EPMC1618307 | biostudies-literature |
REPOSITORIES: biostudies-literature
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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:Detection and quantification of proteins and their post-translational modifications are crucial to decipher functions of complex protein networks in cell biology and medicine. Capillary isoelectric focusing together with antibody-based detection can resolve and identify proteins and their isoforms with modest sample input. However, insufficient sensitivity prevents detection of proteins present at low concentrations and antibody cross-reactivity results in unspecific detection that cannot be distinguished from bona fide protein isoforms. By using DNA-conjugated antibodies enhanced signals can be obtained via rolling circle amplification (RCA). Both sensitivity and specificity can be greatly improved in assays dependent on target recognition by pairs of antibodies using in situ proximity ligation assays (PLA). Here we applied these DNA-assisted RCA techniques in capillary isoelectric focusing to resolve endogenous signaling transducers and isoforms along vascular endothelial growth factor (VEGF) signaling pathways at concentrations too low to be detected in standard assays. We also demonstrate background rejection and enhanced specificity when protein detection depended on binding by pairs of antibodies using in situ PLA, compared to assays where each antibody preparation was used on its own.
| S-EPMC5431457 | biostudies-literature
Project description:Six amino acids with pIs that ranged from 3.2 to 9.7 were used as ampholytes to establish a pH gradient in capillary isoelectric focusing. This amino acid-based capillary isoelectric focusing (cIEF) was coupled with ESI-MS/MS using an electrokinetically pumped sheath-flow interface for peptide analysis. Amino acid-based isoelectric focusing generates a two-order of magnitude lower background signal than commercial ampholytes in the important m/z range of 300-1800. Good focusing was achieved for insulin receptor, which produced ~10 s peak width. For 0.1 mg mL(-1) bovine serum albumin (BSA) digests, 24±1 peptides (sequence coverage 47±4%) were identified in triplicate analysis. As expected, the BSA peptides were separated according to their pI. The concentration detection limit for the BSA digests is 7 nM and the mass detection limit is 7 fmole. A solution of six bovine protein tryptic digests spanning 5 orders of magnitude in concentration was analyzed by amino acid based cIEF-ESI-MS/MS. Five proteins with a concentration range spanning 4 orders of magnitude were identified in triplicate runs. Using amino acid based cIEF-ESI-MS/MS, 112 protein groups and 303 unique peptides were identified in triplicate runs of a RAW 264.7 cell homogenate protein digest. In comparison with ampholyte based cIEF-ESI-MS/MS, amino acid based cIEF-ESI-MS/MS produces higher resolution of five acidic peptides, much cleaner mass spectra, and higher protein spectral counts.
| S-EPMC3474973 | 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:A qualitative, semi-automatized method for apolipoprotein E (apoE) phenotyping by isoelectric focusing method has been evaluated on 40 serum samples from patients previously genotyped for apoE, especially as regards concordance with genotyping, but also repeatability and reproducibility of the method, and sample storage. Total concordance with genotyping and good precision criteria, together with its practicability and requirement of a little sample volume, lead to conclude to the usefulness of this method to help clinicians in the diagnosis of dyslipidemic and neurodegenerative diseases.
| S-EPMC6939034 | 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.
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