RESUMO
Surface interactions are a concern in microscale separations, where analyte adsorption can decrease the speed, sensitivity, and resolution otherwise achieved by miniaturization. Here, we functionally characterize the surface adsorption of hot-embossed cyclic olefin copolymer (COC) micro free-flow electrophoresis (µFFE) devices using two-dimensional nLC × µFFE separations, which introduce a 3- to 5 s plug of analyte into the device and measure temporal broadening that arises from surface interactions. COC is an attractive material for microfluidic devices, but little is known about its potential for surface adsorption in applications with continuous fluid flow and temporal measurements. Adsorption was minimal for three small molecule dyes: positively charged rhodamine 123, negatively charged fluorescein, and neutral rhodamine 110. Temporal peak widths for the three dyes ranged from 3 to 7 s and did not change significantly with increasing transit distance. Moderate adsorption was observed for Chromeo P503-labeled myoglobin and cytochrome c with temporal peak widths around 20 s. Overall, the COC surface adsorption was low compared to traditional glass devices, where peak widths are on the order of minutes. Improvements in durability, long-term performance, and ease of fabrication, combined with low overall adsorption, make the COC µFFE devices a practical choice for applications involving time-resolved continuous detection.
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The fabrication of high-performance microscale devices in substrates with optimal material properties while keeping costs low and maintaining the flexibility to rapidly prototype new designs remains an ongoing challenge in the microfluidics field. To this end, we have fabricated a micro free-flow electrophoresis (µFFE) device in cyclic olefin copolymer (COC) via hot embossing using a PolyJet 3D-printed master mold. A room-temperature cyclohexane vapor bath was used to clarify the device and facilitate solvent-assisted thermal bonding to fully enclose the channels. Device profiling showed 55 µm deep channels with no detectable feature degradation due to solvent exposure. Baseline separation of fluorescein, rhodamine 110, and rhodamine 123, was achieved at 150 V. Limits of detection for these fluorophores were 2 nM, 1 nM, and 10 nM, respectively, and were comparable to previously reported values for glass and 3D-printed devices. Using PolyJet 3D printing in conjunction with hot embossing, the full design cycle, from initial design to production of fully functional COC µFFE devices, could be completed in as little as 6 days without the need for specialized clean room facilities. Replicate COC µFFE devices could be produced from an existing embossing mold in as little as two hours.
RESUMO
We have 3D printed and fabricated micro free-flow electrophoresis (µFFE) devices in acrylonitrile butadiene styrene (ABS) that exhibit minimal surface adsorption without requiring additional surface coatings or specialized buffer additives. 2D, nano LC-micro free flow electrophoresis (2D nLC × µFFE) separations were used to assess both spatial and temporal broadening as peaks eluted through the separation channel. Minimal broadening due to wall adsorption was observed in either the spatial or temporal dimensions during separations of rhodamine 110, rhodamine 123, and fluorescein. Surface adsorption was observed in separations of Chromeo P503 labeled myoglobin and cytochrome c but was significantly reduced compared to previously reported glass devices. Peak widths of < 30 s were observed for both proteins. For comparison, Chromeo P503 labeled myoglobin and cytochrome c adsorb strongly to the surface of glass µFFE devices resulting in peak widths >20 min. A 2D nLC × µFFE separation of a Chromeo P503 labeled tryptic digest of BSA was performed to demonstrate the high peak capacity possible due to the low surface adsorption in the 3D printed ABS devices, even in the absence of surface coatings or buffer additives.
Assuntos
Acrilonitrila/química , Butadienos/química , Eletroforese/instrumentação , Impressão Tridimensional , Estireno/química , Adsorção , Desenho de Equipamento , Corantes Fluorescentes/análise , Corantes Fluorescentes/química , Corantes Fluorescentes/isolamento & purificação , Modelos Químicos , Propriedades de SuperfícieRESUMO
Nucleic acid - protein interactions are critical for regulating gene activation in the nucleus. In the cytoplasm, however, potential nucleic acid-protein functional interactions are less clear. The emergence of a large and expanding number of non-coding RNAs and DNA fragments raises the possibility that the cytoplasmic nucleic acids may interact with cytoplasmic cellular components to directly alter key biological processes within the cell. We now show that both natural and synthetic nucleic acids, collectively XNAs, when introduced to the cytoplasm of live cell cardiac myocytes, markedly enhance contractile function via a mechanism that is independent of new translation, activation of the TLR-9 pathway or by altered intracellular Ca2+ cycling. Findings show a steep XNA oligo length-dependence, but not sequence dependence or nucleic acid moiety dependence, for cytoplasmic XNAs to hasten myocyte relaxation. XNAs localized to the sarcomere in a striated pattern and bound the cardiac troponin regulatory complex with high affinity in an electrostatic-dependent manner. Mechanistically, XNAs phenocopy PKA-based modified troponin to cause faster relaxation. Collectively, these data support a new role for cytoplasmic nucleic acids in directly modulating live cell cardiac performance and raise the possibility that cytoplasmic nucleic acid - protein interactions may alter functionally relevant pathways in other cell types.
Assuntos
Cálcio/metabolismo , Citoplasma/metabolismo , DNA/metabolismo , Miócitos Cardíacos/metabolismo , RNA não Traduzido/metabolismo , Sarcômeros/metabolismo , Animais , Contração Miocárdica , Miócitos Cardíacos/citologia , RatosRESUMO
Tics and compulsions in comorbid Tourette's syndrome (TS) and obsessive-compulsive disorder (OCD) are associated with chronic hyperactivity of parallel cortico/amygdalo-striato-thalamo-cortical (CSTC) loop circuits. Comorbid TS- & OCD-like behaviors have likewise been observed in D1CT-7 mice, in which an artificial neuropotentiating transgene encoding the cAMP-elevating intracellular subunit of cholera toxin (CT) is chronically expressed selectively in somatosensory cortical & amygdalar dopamine (DA) D1 receptor-expressing neurons that activate cortico/amygdalo-striatal glutamate (GLU) output. We've now examined in D1CT-7 mice whether the chronic GLU output from their potentiated cortical/limbic CSTC subcircuit afferents associated with TS- & OCD-like behaviors elicits desensitizing neurochemical changes in the striatum (STR). Microdialysis-capillary electrophoresis and in situ hybridization reveal that the mice's chronic GLU-excited STR exhibits pharmacodynamic changes in three independently GLU-regulated measures of output neuron activation, co-excitation, and desensitization, signifying hyperactive striatal CSTC output and compensatory striatal glial and neuronal desensitization: 1) Striatal GABA, an output neurotransmitter induced by afferent GLU, is increased. 2) Striatal d-serine, a glial excitatory co-transmitter inhibited by afferent GLU, is decreased. 3) Striatal Period1 (Per1), which plays a non-circadian role in the STR as a GLUâ¯+â¯DA D1- (cAMP-) dependent repressor thought to feedback-inhibit GLUâ¯+â¯DA- triggered ultradian urges and motions, is transcriptionally abolished. These data imply that chronic cortical/limbic GLU excitation of the STR desensitizes its co-excitatory d-serine & DA inputs while freezing its GABA output in an active state to mediate chronic tics and compulsions - possibly in part by abolishing striatal Per1-dependent ultradian extinction of urges and motions.
Assuntos
Biomarcadores/análise , Encéfalo/fisiopatologia , Transtorno Obsessivo-Compulsivo/fisiopatologia , Síndrome de Tourette/fisiopatologia , Animais , Encéfalo/metabolismo , Modelos Animais de Doenças , Glutamina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Transtorno Obsessivo-Compulsivo/metabolismo , Síndrome de Tourette/metabolismoRESUMO
Nucleic acid aptamers have tremendous potential as molecular recognition elements in biomedical targeting, analytical arrays, and self-signaling sensors. However, practical limitations and inefficiencies in the process of selecting novel aptamers (SELEX) have hampered widespread adoption of aptamer technologies. Many factors have recently contributed to more effective aptamer screening, but no influence has done more to increase the efficiency, scale, and automation of aptamer selection than that of new microfluidic SELEX techniques. This review introduces aptamers as a powerful chemical and biological tool, briefly highlights traditional SELEX techniques and their limitations, covers in detail the recent advancements in microfluidic methods of aptamer selection and characterization, and suggests possible future directions of the field.
RESUMO
Micro free-flow electrophoresis (µFFE) is a continuous separation technique in which analytes are streamed through a perpendicularly applied electric field in a planar separation channel. Analyte streams are deflected laterally based on their electrophoretic mobilities as they flow through the separation channel. A number of µFFE separation modes have been demonstrated, including free zone (FZ), micellar electrokinetic chromatography (MEKC), isoelectric focusing (IEF) and isotachophoresis (ITP). Approximately 60 articles have been published since the first µFFE device was fabricated in 1994. We anticipate that recent advances in device design, detection, and fabrication, will allow µFFE to be applied to a much wider range of applications. Applications particularly well suited for µFFE analysis include continuous, real time monitoring and microscale purifications.
Assuntos
Eletroforese , Técnicas Analíticas Microfluídicas , Linhagem Celular , Desenho de Equipamento , Humanos , Dispositivos Lab-On-A-Chip , Proteínas/análise , Proteínas/química , Proteínas/isolamento & purificaçãoRESUMO
We have developed an online, high-throughput, microdialysis-capillary electrophoresis (MD-CE) assay for measuring the in vivo dynamics of amino acid biomarkers of metabolism in adipose tissue. Microdialysis probes were implanted into the inguinal adipose tissue depot of C57BL6 mice. The probe location and integrity were verified following each experiment, demonstrating our ability to accurately target the inguinal adipose tissue depot without damaging the probe. The relative concentrations of small molecule bioamines were measured in adipose tissue every 22 s. Arginine, lysine, isoleucine, leucine, methionine, phenylalanine, valine, GABA, glutamine, alanine, glycine and taurine were separated and detected at concentrations significantly higher than the assay LOD. The relative abundances of these analytes were found to be reproducible between mice. The capability of the online MD-CE assay to record dynamic, in vivo changes was assessed by administering an insulin stimulation via tail vein injection. Valine concentrations increased by 40% in response to insulin exposure, while alanine increased by 46% and taurine increased by 37%. Following the initial increase, the amino acid concentrations remained significantly elevated for an extended period (p < 0.001).
Assuntos
Tecido Adiposo/química , Aminoácidos/análise , Internet , Tecido Adiposo/metabolismo , Aminoácidos/metabolismo , Animais , Biomarcadores/análise , Biomarcadores/metabolismo , Eletroforese Capilar , Ensaios de Triagem em Larga Escala , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicrodiáliseRESUMO
Two-dimensional (2D) separations are able to generate significantly higher peak capacities than their one-dimensional counterparts. Unfortunately, current hyphenated 2D separations are limited by the speed of the second dimension separation and the consequent loss of peak capacity due to under sampling of peaks as they elute from the first dimension separation. Continuous micro free flow electrophoresis (µFFE) separations eliminate under sampling as a limitation when incorporated as the second dimension of a 2D separation. In the current manuscript we describe the first coupling of capillary electrophoresis (CE) with µFFE to perform 2D CE × µFFE separations. The CE separation capillary was directly inserted into the µFFE separation channel using an edge on interface. Analyte peaks streamed directly into the µFFE separation channel as they migrated off the CE capillary. No complicated injection, valving, or voltage changes were necessary to couple the two separation modes. 2D CE × µFFE generated an ideal peak capacity of 2â¯592 in a 9 min separation of fluorescently labeled peptides (7.6 min separation window, 342 peaks/min). Data points were recorded every 250-500 ms (>8 data points/peak), effectively eliminating under sampling as a source of band broadening. CE × µFFE generated an ideal peak capacity of 1885 in a 2.7 min separation of fluorescently labeled small molecule bioamines (1.8 min separation window, 1053 peaks/min). Peaks in the 2D CE × µFFE separation of peptides covered 30% of the available separation space, resulting in a corrected peak capacity of 778 (102 peaks/min). The fractional coverage of the 2D CE × µFFE separation of small molecule bioamines was 20%, resulting in a corrected peak capacity of 377 (209 peaks/min).
Assuntos
Aminas/análise , Eletroforese Capilar/métodos , Peptídeos/análise , Aminas/isolamento & purificação , Animais , Bovinos , Peptídeos/isolamento & purificação , Soroalbumina Bovina/química , Soroalbumina Bovina/metabolismoRESUMO
The sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) and phospholamban (PLN) complex regulates heart relaxation through its removal of cytosolic Ca2+ during diastole. Dysfunction of this complex has been related to many heart disorders and is therefore a key pharmacological target. There are currently no therapeutics that directly target either SERCA or PLN. It has been previously reported that single-stranded DNA binds PLN with strong affinity and relieves inhibition of SERCA in a length-dependent manner. In the current article, we demonstrate that RNAs and single-stranded oligonucleotide analogs, or xeno nucleic acids (XNAs), also bind PLN strongly (Kd <10 nm) and relieve inhibition of SERCA. Affinity for PLN is sequence-independent. Relief of PLN inhibition is length-dependent, allowing SERCA activity to be restored incrementally. The improved in vivo stability of XNAs offers more realistic pharmacological potential than DNA or RNA. We also found that microRNAs (miRNAs) 1 and 21 bind PLN strongly and relieve PLN inhibition of SERCA to a greater extent than a similar length random sequence RNA mixture. This may suggest that miR-1 and miR-21 have evolved to contain distinct sequence elements that are more effective at relieving PLN inhibition than random sequences.
Assuntos
Proteínas de Ligação ao Cálcio/química , MicroRNAs/química , Oligonucleotídeos/química , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/química , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Oligonucleotídeos/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismoRESUMO
We have developed a high-throughput microdialysis-capillary electrophoresis (MD-CE) assay for monitoring branched chain amino acid (BCAA) uptake/release dynamics in 3T3-L1 cells. BCAAs (i.e., isoleucine, leucine, and valine) and their downstream metabolites (i.e., alanine, glutamine, and glutamate) are important indicators of adipocyte lipogenesis. To perform an analysis, amino acids were sampled using microdialysis, fluorescently labeled in an online reaction, separated using CE, and detected using laser-induced fluorescence (LIF) in a sheath flow cuvette. Separation conditions were optimized for the resolution of the BCAAs isoleucine, leucine, and valine, as well as 13 other amino acids, including ornithine, alanine, glutamine, and glutamate. CE separations were performed in <30 s, and the temporal resolution of the online MD-CE assay was <60 s. Limits of detection (LOD) were 400, 200, and 100 nM for isoleucine, leucine, and valine, respectively. MD-CE dramatically improved throughput in comparison to traditional offline CE methods, allowing 8 replicates of 15 samples (i.e., 120 analyses) to be assayed in <120 min. The MD-CE assay was used to assess the metabolism dynamics of 3T3-L1 cells over time, confirming the utility of the assay.
Assuntos
Aminoácidos de Cadeia Ramificada/análise , Eletroforese Capilar/métodos , Células 3T3-L1 , Aminoácidos de Cadeia Ramificada/química , Animais , Eletroforese Capilar/instrumentação , Corantes Fluorescentes/química , Ácido Glutâmico/análise , Ácido Glutâmico/química , Isoleucina/análise , Isoleucina/química , Leucina/análise , Leucina/química , Limite de Detecção , Camundongos , MicrodiáliseRESUMO
The cost, time, and restrictions on creative flexibility associated with current fabrication methods present significant challenges in the development and application of microfluidic devices. Additive manufacturing, also referred to as three-dimensional (3D) printing, provides many advantages over existing methods. With 3D printing, devices can be made in a cost-effective manner with the ability to rapidly prototype new designs. We have fabricated a micro free-flow electrophoresis (µFFE) device using a low-cost, consumer-grade 3D printer. Test prints were performed to determine the minimum feature sizes that could be reproducibly produced using 3D printing fabrication. Microfluidic ridges could be fabricated with dimensions as small as 20 µm high × 640 µm wide. Minimum valley dimensions were 30 µm wide × 130 µm wide. An acetone vapor bath was used to smooth acrylonitrile-butadiene-styrene (ABS) surfaces and facilitate bonding of fully enclosed channels. The surfaces of the 3D-printed features were profiled and compared to a similar device fabricated in a glass substrate. Stable stream profiles were obtained in a 3D-printed µFFE device. Separations of fluorescent dyes in the 3D-printed device and its glass counterpart were comparable. A µFFE separation of myoglobin and cytochrome c was also demonstrated on a 3D-printed device. Limits of detection for rhodamine 110 were determined to be 2 and 0.3 nM for the 3D-printed and glass devices, respectively.
Assuntos
Citocromos c/isolamento & purificação , Eletroforese/métodos , Mioglobina/isolamento & purificação , Impressão Tridimensional/instrumentação , Eletroforese/economia , Eletroforese/instrumentação , Limite de Detecção , Microfluídica , Rodaminas/análiseRESUMO
Multidimensional separations present a unique opportunity for generating the high peak capacities necessary for the analysis of complex biological mixtures. We have coupled nano liquid chromatography with micro free flow electrophoresis (nLC × µFFE) to produce high peak capacity separations of peptide and amino acid mixtures. Currently, µFFE largely relies on laser-induced fluorescence (LIF) detection. We have demonstrated that the choice of fluorescent label significantly affects the fractional coverage and peak capacity of nLC × µFFE separations of peptides and amino acids. Of the labeling reagents assessed, Chromeo P503 performed the best for nLC × µFFE separations of peptides. A nLC × µFFE analysis of a Chromeo P503-labeled BSA tryptic digest produced a 2D separation that made effective use of the available separation space (48%), generating a corrected peak capacity of 521 in a 5 min separation window (104 peaks/min). nLC × µFFE separations of NBD-F-labeled peptides produced similar fractional coverage and peak capacity, but this reagent was able to react with multiple reaction sites, producing an unnecessarily complex analyte mixture. NBD-F performed the best for nLC × µFFE separations of amino acids. NBD-F-labeled amino acids produced a 2D separation that covered 36% of the available separation space, generating a corrected peak capacity of 95 in a 75 s separation window (76 peaks/min). Chromeo P503 and Alexa Fluor 488-labeled amino acids were not effectively separated in the µFFE dimension, giving 2D separations with poor fractional coverage and peak capacity.
Assuntos
Aminoácidos/isolamento & purificação , Fluorescência , Nanotecnologia , Peptídeos/isolamento & purificação , Cromatografia Líquida/instrumentação , Eletroforese/instrumentação , Lasers , Nanotecnologia/instrumentaçãoRESUMO
Analyte adsorption onto surfaces presents a challenge for many separations, often becoming a significant source of peak broadening and asymmetry. We have shown that surface adsorption has no effect on peak position or spatial broadening in micro free flow electrophoresis (µFFE) separations. Surface adsorption does affect the time it takes an analyte to travel through the µFFE separation channel and therefore contributes to temporal broadening. These results were confirmed using µFFE separations of fluorescein, rhodamine 110, and rhodamine 123 in a low ionic strength buffer to promote surface adsorption. Peak widths and asymmetries were measured in both the temporal and spatial dimensions. Under these conditions rhodamine 123 exhibited significant interactions with the separation channel surface, causing increased peak broadening and asymmetry in the temporal dimension. Broadening or asymmetry in the spatial dimension was not significantly different than that of fluorescein, which did not interact with the capillary surface. The effect of strong surface interactions was assessed using µFFE separations of Chromeo P503 labeled myoglobin and cytochrome c. Myoglobin and cytochrome c were well resolved and gave rise to symmetrical peaks in the spatial dimension even under conditions where permanent adsorption onto the separation channel surface occurred.
Assuntos
Eletroforese , Adsorção , Citocromos c/química , Citocromos c/isolamento & purificação , Eletroforese/instrumentação , Fluoresceína/química , Fluoresceína/isolamento & purificação , Mioglobina/química , Mioglobina/isolamento & purificação , Concentração Osmolar , Tamanho da Partícula , Rodamina 123/química , Rodamina 123/isolamento & purificação , Rodaminas/química , Rodaminas/isolamento & purificação , Propriedades de Superfície , Fatores de TempoRESUMO
The membrane protein complex between sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) and phospholamban (PLN) is a prime therapeutic target for reversing cardiac contractile dysfunctions caused by calcium mishandling. So far, however, efforts to develop drugs specific for this protein complex have failed. Here, we show that non-coding RNAs and single-stranded DNAs (ssDNAs) interact with and regulate the function of the SERCA/PLN complex in a tunable manner. Both in HEK cells expressing the SERCA/PLN complex, as well as in cardiac sarcoplasmic reticulum preparations, these short oligonucleotides bind and reverse PLN's inhibitory effects on SERCA, increasing the ATPase's apparent Ca(2+) affinity. Solid-state NMR experiments revealed that ssDNA interacts with PLN specifically, shifting the conformational equilibrium of the SERCA/PLN complex from an inhibitory to a non-inhibitory state. Importantly, we achieved rheostatic control of SERCA function by modulating the length of ssDNAs. Since restoration of Ca(2+) flux to physiological levels represents a viable therapeutic avenue for cardiomyopathies, our results suggest that oligonucleotide-based drugs could be used to fine-tune SERCA function to counterbalance the extent of the pathological insults.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , DNA de Cadeia Simples/metabolismo , Proteínas de Membrana/metabolismo , Oligonucleotídeos/metabolismo , RNA não Traduzido/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Aminoácidos/metabolismo , Animais , Sobrevivência Celular , Epitopos/metabolismo , Transferência Ressonante de Energia de Fluorescência , Cinética , Espectroscopia de Ressonância Magnética , Ligação Proteica , Coelhos , Retículo Sarcoplasmático/metabolismo , Sus scrofaRESUMO
The throughput of existing liquid phase two-dimensional separations is generally limited by the peak capacity lost due to under sampling by the second dimension separation as peaks elute off the first dimension separation. In the current manuscript, a first dimension nanoliquid chromatography (nLC) separation is coupled directly with a second dimension micro free flow electrophoresis (µFFE) separation. Since µFFE performs continuous separations, no complicated injection or modulation is necessary to couple the two techniques. Analyte peaks are further separated in µFFE as they elute off the nLC column. A side-on interface was designed to minimize dead volume in the nLC × µFFE interface, eliminating this as a source of band broadening. A Chromeo P503 labeled tryptic digest of BSA was used as a complex mixture to assess peak capacity. 2D nLC × µFFE peak capacities as high as 2,352 could be obtained in a 10 min separation window when determined according to the product of the first and second dimension peak capacities. After considering the orthogonality of the two separation modes and the fraction of separation space occupied by peaks, the usable peak capacity generated was determined to be 776. The 105 peaks/min generated using 2D nLC × µFFE was nearly double the previously reported maximum peak capacity production rate achieved using online LC × LC.
Assuntos
Nanotecnologia/métodos , Peptídeos/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Eletroforese , Hidrolisados de Proteína/química , Soroalbumina Bovina/química , TripsinaRESUMO
Various isoforms of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) regulate Ca2+ homeostatic balance in both the heart (SERCA2a) and skeletal muscle (SERCA1a). Ca2+ plays a key role in these tissues as an intracellular signal that controls contractility. Due to its key role in the contractility cycle, SERCA is emerging as a promising pharmacological target to modulate heart muscle function. SERCA function is regulated by its endogenous inhibitor phospholamban (PLN). Upon binding, PLN decreases SERCA's apparent affinity for Ca2+. Therefore the interaction between PLN and SERCA has an important role in determining both physiological and pathological conditions. Quantifying the inhibitory potency of PLN is of great importance in understanding the pathophysiology of heart muscle. Traditionally, SERCA activity assays have been performed using a PK/LDH-coupled enzyme reaction, which suffers from limited sensitivity. We have developed a new SERCA activity assay based on the direct detection of the product ADP via time resolved FRET (TR-FRET). Under optimized conditions, our assay reduced the amount of SERCA required to perform the assay 1,000-fold. Inter-day reproducibility was shown to be excellent for SERCA preparations in either detergent (C12E8) or reconstituted lipids. The inhibitory effect of PLN on SERCA measured under the low-concentration conditions of our assay allowed us to more accurately investigate the binding between PLN and SERCA. Significant inhibitory effects of PLN were observed even at mid-nanomolar concentrations significantly lower than previous Kd estimates for the SERCA-PLN complex.
RESUMO
Mitochondria are responsible for maintaining a variety of cellular functions. One such function is the interaction and subsequent import of proteins into these organelles via the translocase of outer membrane (TOM) complex. Antibodies have been used to analyze the presence and function of proteins comprising this complex, but have not been used to investigate variations in the abundance of TOM complex in mitochondria. Here, we report on the feasibility of using capillary cytometry with laser-induced fluorescence to detect mitochondria labeled with antibodies targeting the TOM complex and to estimate the number of antibodies that bind to these organelles. Mitochondria were fluorescently labeled with DsRed2, while antibodies targeting the TOM22 protein, one of nine proteins comprising the TOM complex, were conjugated to the Atto-488 fluorophore. At typical labeling conditions, 94% of DsRed2 mitochondria were also immunofluorescently labeled with Atto-488 Anti-TOM22 antibodies. The calculated median number of Atto-488 Anti-TOM22 antibodies bound to the surface of mitochondria was â¼2,000 per mitochondrion. The combination of fluorescent immunolabeling and capillary cytometry could be further developed to include multicolor labeling experiments, which enable monitoring several molecular targets at the same time in the same or different organelle types.
Assuntos
Anticorpos/análise , Corantes Fluorescentes/análise , Mitocôndrias/ultraestrutura , Proteínas de Transporte da Membrana Mitocondrial/análise , Linhagem Celular Tumoral , Fluoresceínas/análise , Humanos , Imuno-Histoquímica/métodos , Proteínas Luminescentes/análise , Microscopia Confocal , Mitocôndrias/química , Proteínas do Complexo de Importação de Proteína Precursora MitocondrialRESUMO
Isolating a particular strand of DNA from a double stranded DNA duplex is an important step in aptamer generation as well as many other biotechnology applications. Here we describe a microfluidic, flow-through, dialysis device for isolating single-stranded DNA (ssDNA) from double-stranded DNA (dsDNA). The device consists of two channels fabricated in polydimethylsiloxane (PDMS) separated by a track etched polycarbonate membrane (800 nm pore size). To isolate ssDNA, dual-biotin labelled dsDNA was immobilized onto streptavidin-coated polystyrene beads. Alkaline treatment was used to denature dsDNA, releasing the non-biotinylated ssDNA. In the flow-through dialysis device the liberated ssDNA was able to cross the membrane and was collected in an outlet channel. The complementary sequence bound to the bead was unable to cross the membrane and was directed to a waste channel. The effect of NaOH concentration and flow rate on purity and yield were compared. >95% ssDNA purity was achieved at 25 mM NaOH. However, lower flow rates were necessary to achieve ssDNA yields approaching the 50% theoretical maximum of the concurrent-flow device. Under optimized conditions the microfluidic isolation achieved even higher purity ssDNA than analogous manual procedures.