48 research outputs found
A Microfluidic Device with Integrated Sonication and Immunoprecipitation for Sensitive Epigenetic Assays
Epigenetic
studies increasingly require analysis of a small number
of cells that are of one specific type and derived from patients or
animals. In this report, we demonstrate a simple microfluidic device
that integrates sonication and immunoprecipitation (IP) for epigenetic
assays, such as chromatin immunoprecipitation (ChIP) and methylated
DNA immunoprecipitation (MeDIP). By incorporating an ultrasonic transducer
with a microfluidic chamber, we implemented microscale sonication
for both shearing chromatin/DNA and mixing/washing of IP beads. Such
integration allowed highly sensitive tests starting with 100 cross-linked
cells for ChIP or 500 pg of genomic DNA for MeDIP (compared to 10<sup>6</sup>–10<sup>7</sup> cells for ChIP and 1–10 μg
of DNA for MeDIP in conventional assays). The entire on-chip process
of sonication and IP took only 1 h. Our tool will be useful for highly
sensitive epigenetic studies based on a small quantity of sample
A Microfluidic Device with Integrated Sonication and Immunoprecipitation for Sensitive Epigenetic Assays
Epigenetic
studies increasingly require analysis of a small number
of cells that are of one specific type and derived from patients or
animals. In this report, we demonstrate a simple microfluidic device
that integrates sonication and immunoprecipitation (IP) for epigenetic
assays, such as chromatin immunoprecipitation (ChIP) and methylated
DNA immunoprecipitation (MeDIP). By incorporating an ultrasonic transducer
with a microfluidic chamber, we implemented microscale sonication
for both shearing chromatin/DNA and mixing/washing of IP beads. Such
integration allowed highly sensitive tests starting with 100 cross-linked
cells for ChIP or 500 pg of genomic DNA for MeDIP (compared to 10<sup>6</sup>–10<sup>7</sup> cells for ChIP and 1–10 μg
of DNA for MeDIP in conventional assays). The entire on-chip process
of sonication and IP took only 1 h. Our tool will be useful for highly
sensitive epigenetic studies based on a small quantity of sample
A Microfluidic Device with Integrated Sonication and Immunoprecipitation for Sensitive Epigenetic Assays
Epigenetic
studies increasingly require analysis of a small number
of cells that are of one specific type and derived from patients or
animals. In this report, we demonstrate a simple microfluidic device
that integrates sonication and immunoprecipitation (IP) for epigenetic
assays, such as chromatin immunoprecipitation (ChIP) and methylated
DNA immunoprecipitation (MeDIP). By incorporating an ultrasonic transducer
with a microfluidic chamber, we implemented microscale sonication
for both shearing chromatin/DNA and mixing/washing of IP beads. Such
integration allowed highly sensitive tests starting with 100 cross-linked
cells for ChIP or 500 pg of genomic DNA for MeDIP (compared to 10<sup>6</sup>–10<sup>7</sup> cells for ChIP and 1–10 μg
of DNA for MeDIP in conventional assays). The entire on-chip process
of sonication and IP took only 1 h. Our tool will be useful for highly
sensitive epigenetic studies based on a small quantity of sample
Microfluidics-Based Chromosome Conformation Capture (3C) Technology for Examining Chromatin Organization with a Low Quantity of Cells
Detecting three-dimensional (3D)
genome organization in the form
of physical interactions between various genomic loci is of great
importance for understanding transcriptional regulations and cellular
fate. Chromosome Conformation Capture (3C) method is the gold standard
for examining chromatin organization, but usually requires a large
number of cells (>10<sup>7</sup>). This hinders studies of scarce
tissue samples from animals and patients using the method. Here we
developed a microfluidics-based approach for examining chromosome
conformation by 3C technology. Critical 3C steps, such as digestion
and religation of BAC DNA and cross-linked chromatin, were implemented
on a microfluidic chip using a low quantity of cells (<10<sup>4</sup>). Using this technology, we analyzed the chromatin looping interactions
in the human β-globin. We envision that our method will provide
a powerful tool for low-input analysis of chromosome conformation
and epigenetic regulations
A Microfluidic Device with Integrated Sonication and Immunoprecipitation for Sensitive Epigenetic Assays
Epigenetic
studies increasingly require analysis of a small number
of cells that are of one specific type and derived from patients or
animals. In this report, we demonstrate a simple microfluidic device
that integrates sonication and immunoprecipitation (IP) for epigenetic
assays, such as chromatin immunoprecipitation (ChIP) and methylated
DNA immunoprecipitation (MeDIP). By incorporating an ultrasonic transducer
with a microfluidic chamber, we implemented microscale sonication
for both shearing chromatin/DNA and mixing/washing of IP beads. Such
integration allowed highly sensitive tests starting with 100 cross-linked
cells for ChIP or 500 pg of genomic DNA for MeDIP (compared to 10<sup>6</sup>–10<sup>7</sup> cells for ChIP and 1–10 μg
of DNA for MeDIP in conventional assays). The entire on-chip process
of sonication and IP took only 1 h. Our tool will be useful for highly
sensitive epigenetic studies based on a small quantity of sample
Quantitative Analysis of Ligand Induced Heterodimerization of Two Distinct Receptors
The
induced dimerization of two distinct receptors through a heterobifunctional
inducer is prevalent among all levels of cellular signaling processes,
yet its complexity poses difficulty for systematic quantitative analysis.
This paper first shows how to calculate the amount of any possible
complex or monomer of heteroligand and two receptors present at equilibrium.
The theory is subsequently applied to the determination of three independent
equilibrium parameters involved in the rapamycin induced FKBP and
FRB dimerization, in which all parameters were simultaneously estimated
using one set of fluorescence resonance energy transfer (FRET) experiments.
A MATLAB script is provided for parametric fitting
Additional file 1 of NoGOA: predicting noisy GO annotations using evidences and sparse representation
Supplementary file of ‘NoGOA: predicting noisy GO annotations using evidences and sparse representation’ This PDF file includes additional experimental results mentioned in the main text. (PDF 1300 kb
Distinct Enzyme–Substrate Interactions Revealed by Two Dimensional Kinetic Comparison between Dehaloperoxidase-Hemoglobin and Horseradish Peroxidase
The
time-resolved kinetics of substrate oxidation and cosubstrate
H<sub>2</sub>O<sub>2</sub> reduction by dehaloperoxidase-hemoglobin
(DHP) on a seconds-to-minutes time scale was analyzed for peroxidase
substrates 2,4,6-tribromophenol (2,4,6-TBP), 2,4,6-trichlorophenol
(2,4,6-TCP), and ABTS. Substrates 2,4,6-TBP and 2,4,6-TCP show substrate
inhibition at high concentration due to the internal binding at the
distal pocket of DHP, whereas ABTS does not show substrate inhibition
at any concentration. The data are consistent with an external binding
site for the substrates with an internal substrate inhibitor binding
site for 2,4,6-TBP and 2,4,6-TCP. We have also compared the kinetic
behavior of horseradish peroxidase (HRP) in terms of <i>k</i><sub>cat</sub>, <i>K</i><sub>m</sub><sup>AH<sub>2</sub></sup> and <i>K</i><sub>m</sub><sup>H<sub>2</sub>O<sub>2</sub></sup> using the same kinetic scheme. Unlike DHP, HRP does not exhibit
any measurable substrate inhibition, consistent with substrate binding
at the edge of heme near the protein surface at all substrate concentrations.
The binding of substrates and their interactions with the heme iron
were further compared between DHP and HRP using a competitive fluoride
binding experiment, which provides a method for quantitative measurement
of internal association constants associated with substrate inhibition.
These experiments show the regulatory role of an internal substrate
binding site in DHP from both a kinetic and competitive ligand binding
perspective. The interaction of DHP with substrates as a result of
internal binding actually stabilizes that protein and permits DHP
to function under conditions that denature HRP. As a consequence,
DHP is a tortoise, a slow but steady enzyme that wins the evolutionary
race against the HRP-type of peroxidase, which is a hare, initially
rapid, but flawed for this application because of the protein denaturation
under the conditions of the experiment
Intracellular Tracking of Single Native Molecules with Electroporation-Delivered Quantum Dots
Quantum dots (QDs) have found a wide
range of biological applications
as fluorophores due to their extraordinary brightness and high photostability
that are far superior to those of conventional organic dyes. These
traits are particularly appealing for studying cell biology under
a cellular autofluorescence background and with a long observation
period. However, it remains the most important open challenge to target
QDs at <i>native</i> intracellular molecules and organelles
in <i>live</i> cells. Endocytosis-based delivery methods
lead to QDs encapsulated in vesicles that have their surface biorecognition
element hidden from the intracellular environment. The probing of
native molecules using QDs has been seriously hindered by the lack
of consistent approaches for delivery of QDs with exposed surface
groups. In this study, we demonstrate that electroporation (i.e.,
the application of short electric pulses for cell permeabilization)
generates reproducible results for delivering QDs into cells. We show
evidence that electroporation-based delivery does not involve endocytosis
or vesicle encapsulation of QDs. The amount of QD loading and the
resulting cell viability can be adjusted by varying the parameters
associated with the electroporation operation. To demonstrate the
application of our approach for intracellular targeting, we study
single-molecule motility of kinesin in live cells by labeling native
kinesins using electroporation-delivered QDs. We envision that electroporation
may serve as a simple and universal tool for delivering QDs into cells
to label and probe native molecules and organelles
Intracellular Tracking of Single Native Molecules with Electroporation-Delivered Quantum Dots
Quantum dots (QDs) have found a wide
range of biological applications
as fluorophores due to their extraordinary brightness and high photostability
that are far superior to those of conventional organic dyes. These
traits are particularly appealing for studying cell biology under
a cellular autofluorescence background and with a long observation
period. However, it remains the most important open challenge to target
QDs at <i>native</i> intracellular molecules and organelles
in <i>live</i> cells. Endocytosis-based delivery methods
lead to QDs encapsulated in vesicles that have their surface biorecognition
element hidden from the intracellular environment. The probing of
native molecules using QDs has been seriously hindered by the lack
of consistent approaches for delivery of QDs with exposed surface
groups. In this study, we demonstrate that electroporation (i.e.,
the application of short electric pulses for cell permeabilization)
generates reproducible results for delivering QDs into cells. We show
evidence that electroporation-based delivery does not involve endocytosis
or vesicle encapsulation of QDs. The amount of QD loading and the
resulting cell viability can be adjusted by varying the parameters
associated with the electroporation operation. To demonstrate the
application of our approach for intracellular targeting, we study
single-molecule motility of kinesin in live cells by labeling native
kinesins using electroporation-delivered QDs. We envision that electroporation
may serve as a simple and universal tool for delivering QDs into cells
to label and probe native molecules and organelles