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Quantification of Protein Interaction Network Dynamics using Multiplexed Co-Immunoprecipitation.

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic interactions among multiple proteins in a protein interaction network is technically difficult. Here, we present a protocol for Quantitative Multiplex Immunoprecipitation (QMI), which allows quantitative assessment of fold changes in protein interactions based on relative fluorescence measurements of Proteins in Shared Complexes detected by Exposed Surface epitopes (PiSCES). In QMI, protein complexes from cell lysates are immunoprecipitated onto microspheres, and then probed with a labeled antibody for a different protein in order to quantify the abundance of PiSCES. Immunoprecipitation antibodies are conjugated to different MagBead spectral regions, which allows a flow cytometer to differentiate multiple parallel immunoprecipitations and simultaneously quantify the amount of probe antibody associated with each. QMI does not require genetic tagging and can be performed using minimal biomaterial compared to other immunoprecipitation methods. QMI can be adapted for any defined group of interacting proteins, and has thus far been used to characterize signaling networks in T cells and neuronal glutamate synapses. Results have led to new hypothesis generation with potential diagnostic and therapeutic applications. This protocol includes instructions to perform QMI, from the initial antibody panel selection through to running assays and analyzing data. The initial assembly of a QMI assay involves screening antibodies to generate a panel, and empirically determining an appropriate lysis buffer. The subsequent reagent preparation includes covalently coupling immunoprecipitation antibodies to MagBeads, and biotinylating probe antibodies so they can be labeled by a streptavidin-conjugated fluorophore. To run the assay, lysate is mixed with MagBeads overnight, and then beads are divided and incubated with different probe antibodies, and then a fluorophore label, and read by flow cytometry. Two statistical tests are performed to identify PiSCES that differ significantly between experimental conditions, and results are visualized using heatmaps or node-edge diagrams.

1934 related Products with: Quantification of Protein Interaction Network Dynamics using Multiplexed Co-Immunoprecipitation.

AKT1 & CDKN1A Protein Pro FAS & DAXX Protein Protei TGFA & ERBB2 Protein Prot HDAC2 & HIF1A Protein Pro PRKCZ & GRM5 Protein Prot CRK & MAPK8 Protein Prote STAT5B & STAT3 Protein Pr CDC6 & MCM7 Protein Prote FGFR1 & CDH1 Protein Prot IKBKB & CHUK Protein Prot KIT & BCR Protein Protein PRKCZ & F11R Protein Prot

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Single-Cell Point Constrictions for Reagent-Free High-Throughput Mechanical Lysis and Intact Nuclei Isolation.

Highly localized (point) constrictions featuring a round geometry with ultra-sharp edges in silicon have been demonstrated for the reagent-free continuous-flow rapid mechanical lysis of mammalian cells on a single-cell basis. Silicon point constrictions, robust structures formed by a single-step dry etching process, are arranged in a cascade along microfluidic channels and can effectively rupture cells delivered in a pressure-driven flow. The influence of the constriction size and count on the lysis performance is presented for fibroblasts in reference to total protein, DNA, and intact nuclei levels in the lysates evaluated by biochemical and fluoremetric assays and flow-cytometric analyses. Protein and DNA levels obtained from an eight-constriction treatment match or surpass those from a chemical method. More importantly, many intact nuclei are found in the lysates with a relatively high nuclei-isolation efficiency from a four-constriction treatment. Point constrictions and their role in rapid reagent-free disruption of the plasma membrane could have implications for integrated sample preparation in future lab-on-a-chip systems.

1605 related Products with: Single-Cell Point Constrictions for Reagent-Free High-Throughput Mechanical Lysis and Intact Nuclei Isolation.

ProPrep™ Omni 200 High- DirectPCR Lysis Reagent ( ProPrep™ BAC 960 High-T ProPrep™ Genomic XL-10 DirectPCR Lysis Reagent ( DNA kits, Column-free Iso Cellufine Formyl , 50 ml AccuzolTM Total RNA Extra Nucleic Acid Isolation En Apoptotic Cell Isolation Cell Lysis Buffer Cell Tissue Lysis Buffer

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Intraoperative tumor lysis syndrome in a giant teratoma: a case report.

Tumor lysis syndrome is an unusual metabolic emergency in solid tumors. Perioperative occurrence of this syndrome is extremely rare but may have fatal consequences if not detected and treated on time.

2400 related Products with: Intraoperative tumor lysis syndrome in a giant teratoma: a case report.

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Evaluation of automated erythrocyte methodology in new world camelids using the ADVIA 2120 hematology analyzer.

Accurate erythrocyte measurements with ADVIA hematology analyzers require isovolumetric cell sphering in one reaction and hemolysis in another. However, camelid erythrocytes are resistant to sphering and osmotic lysis, and no published evaluation of ADVIA methods for camelids exists.

1758 related Products with: Evaluation of automated erythrocyte methodology in new world camelids using the ADVIA 2120 hematology analyzer.

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Efficient synchronization of Plasmodium knowlesi in vitro cultures using guanidine hydrochloride.

Long-term in vitro culture of blood stage Plasmodium parasites invariably leads to asynchronous parasite development. The most often used technique to synchronize Plasmodium falciparum culture is sorbitol treatment, which differentially induces osmotic lysis of trophozoite- and schizont-infected red blood cells due to presence of the new permeation pathways in the membranes of these cells. However, sorbitol treatment does not work well when used to synchronize the culture-adapted Plasmodium knowlesi A1-H.1 line.

2082 related Products with: Efficient synchronization of Plasmodium knowlesi in vitro cultures using guanidine hydrochloride.

D,L-7-Aza-3-indolylglycin N-[2-[(6-Amino-5-nitro-2- Transfection Reagents and Human integrin aVb3, affi Guanidine hydrochloride C Inhibitory Mouse Monoclon Cultrex In Vitro Angiogen Guanidine hydrochloride C Mouse Anti-Plasmodium fal Guanidine hydrochloride C Inhibitory Mouse Monoclon Resorufin Oleate, Fluorog

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A sample processing method for immunoassay of whole blood tacrolimus.

Current sample processing (SP) methods for tacrolimus (FK506) immunoassays are mainly based on extraction of drug by organic solvent and divalent metal ions. Although these methods are effective for drug extraction and interference elimination, they suffer from drawbacks including inconvenience for operation, difficulties for automation and potential measurement bias. To overcome these limitations, this study describes a new SP reagent for blood cell lysis and protein denaturation. A TRFIA (time-resolved fluorescence immunoassay) was developed by using this SP reagent for whole blood FK506 quantification. Results show that blood samples could be turned into homogeneous solution after being treated by this SP reagent, and so could be directly applied to immunoassays without centrifugation. The analytical sensitivity of the FK506-TRFIA was 0.57 ng/mL, the within-run and between-run coefficient of variations (CVs) were both less than 10%. The FK506 values of 126 samples obtained by FK506-TRFIA correlated excellently with that obtained by ABBOTT FK506-CMIA (R = 0.982). Comparison studies also show that the FK506-TRFIA was highly resistant to endogenous interferences. These results suggest that the present SP method is a more promising chose for FK506 immunoassay, and in the meantime, its simplicity makes the whole-process immunoassay automation more feasible by obviating the necessary for centrifugation.

2833 related Products with: A sample processing method for immunoassay of whole blood tacrolimus.

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The effect of the antisickling compound GBT1118 on the permeability of red blood cells from patients with sickle cell anemia.

Sickle cell anemia (SCA) is one of the commonest severe inherited disorders. Nevertheless, effective treatments remain inadequate and novel ones are avidly sought. A promising advance has been the design of novel compounds which react with hemoglobin S (HbS) to increase oxygen (O ) affinity and reduce sickling. One of these, voxelotor (GBT440), is currently in advanced clinical trials. A structural analogue, GBT1118, was investigated in the current work. As RBC dehydration is important in pathogenesis of SCA, the effect of GBT1118 on RBC cation permeability was also studied. Activities of P , the Gardos channel and the KCl cotransporter (KCC) were all reduced. Gardos channel and KCC activities were also inhibited in RBCs treated with Ca ionophore or the thiol reagent N-ethylmaleimide, indicative of direct effects on these two transport systems. Consistent with its action on RBC membrane transporters, GBT1118 significantly increased RBC hydration. RBC hemolysis was reduced in a nonelectrolyte lysis assay. Further to its direct effects on O affinity, GBT1118 was therefore found to reduce RBC shrinkage and fragility. Findings reveal important effects of GBT1118 on protecting sickle cells and suggest that this is approach may represent a useful therapy for amelioration of the clinical complications of SCA.

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Tissue array of ovarian g Guinea Pig Red Blood Cell Mouse Red Blood Cells 10m Recombinant Thermostable Canine Red Blood Cells 10 Rabbit Anti-Theophylline TCP-1 theta antibody Sour Sheep Red Blood Cells, Pa FDA Standard Frozen Tissu Rabbit anti PKC theta (pS Horse Red Blood Cells, Pa ELISA TEK™ MBM Thermal

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Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology.

Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness the transcriptional and translational capacity of cells within a test tube. By obviating the need to maintain the viability of the cell, and by eliminating the cellular barrier, CFPS has been foundational to emerging applications in biomanufacturing of traditionally challenging proteins, as well as applications in rapid prototyping for metabolic engineering, and functional genomics. Our methods for implementing an E. coli-based CFPS platform allow new users to access many of these applications. Here, we describe methods to prepare extract through the use of enriched media, baffled flasks, and a reproducible method of tunable sonication-based cell lysis. This extract can then be used for protein expression capable of producing 900 µg/mL or more of super folder green fluorescent protein (sfGFP) in just 5 h from experimental setup to data analysis, given that appropriate reagent stocks have been prepared beforehand. The estimated startup cost of obtaining reagents is $4,500 which will sustain thousands of reactions at an estimated cost of $0.021 per µg of protein produced or $0.019 per µL of reaction. Additionally, the protein expression methods mirror the ease of the reaction setup seen in commercially available systems due to optimization of reagent pre-mixes, at a fraction of the cost. In order to enable the user to leverage the flexible nature of the CFPS platform for broad applications, we have identified a variety of aspects of the platform that can be tuned and optimized depending on the resources available and the protein expression outcomes desired.

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AccuRapid™ Cell Free Pr Bone Morphogenetic Protei B-cell linker protein ant Rabbit Anti-Cell death in Rabbit Anti-Cell death in Recombinant Human AACT SE Recombinant Human Inhibin Anti-daf-2(Abnormal dauer Mouse Anti-Escherichia co Rabbit Anti-Cell death in Rabbit Anti-Cell death in cell cycle progression 2

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A diffusion-based microfluidic device for single-cell RNA-seq.

Microfluidic devices provide a low-input and efficient platform for single-cell RNA-seq (scRNA-seq). Existing microfluidic devices have a complicated multi-chambered structure for handling the multi-step process involved in RNA-seq and dilution between steps is used to negate the inhibitory effects among reagents. This makes the device difficult to fabricate and operate. Here we present microfluidic diffusion-based RNA-seq (MID-RNA-seq) for conducting scRNA-seq with a diffusion-based reagent swapping scheme. This device incorporates cell trapping, lysis, reverse transcription and PCR amplification all in one simple microfluidic device. MID-RNA-seq provides high data quality that is comparable to existing scRNA-seq methods while implementing a simple device design that permits multiplexing. The robustness and scalability of the MID-RNA-seq device will be important for transcriptomic studies of scarce cell samples.

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Integration of marker-free selection of single cells at a wireless electrode array with parallel fluidic isolation and electrical lysis.

We present integration of selective single-cell capture at an array of wireless electrodes (bipolar electrodes, BPEs) with transfer into chambers, reagent exchange, fluidic isolation and rapid electrical lysis in a single platform, thus minimizing sample loss and manual intervention steps. The whole process is achieved simply by exchanging the solution in a single inlet reservoir and by adjusting the applied voltage at a pair of driving electrodes, thus making this approach particularly well-suited for a broad range of research and clinical applications. Further, the use of BPEs allows the array to be scalable to increase throughput. Specific innovations reported here include the incorporation of a leak channel to balance competing flow paths, the use of 'split BPEs' to create a distinct recapture and electrical lysis point within the reaction chamber, and the dual purposing of an ionic liquid as an immiscible phase to seal the chambers and as a conductive medium to permit electrical lysis at the split BPEs.

2840 related Products with: Integration of marker-free selection of single cells at a wireless electrode array with parallel fluidic isolation and electrical lysis.

Atherosclerosis (Human) A Breast cancer tissue arra Disease State Samples: Si Single Donor Human Atopic Anti apoptotic marker in Mouse Atherosclerosis Arr Esophageal inflammation, Atherosclerosis (Mouse) A Lysis Buffer Antibody Arr Normal human tissue panel Single Donor Human Atopic Anti-apoptotic marker in

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