scholarly journals Parallel sample processing using dispersive INtip micro-purification on programmable multichannel pipettes

BioTechniques ◽  
2020 ◽  
Vol 68 (3) ◽  
pp. 148-154
Author(s):  
Patrick A Kates ◽  
John J Tomashek ◽  
David A Miles ◽  
L Andrew Lee
Keyword(s):  
High Throughput ◽  
Solid Phase ◽  
Affinity Purification ◽  
Manual Intervention ◽  
Parallel Sample ◽  
Spin Column ◽  
Pipette Tip ◽  
Automated Platform ◽  
Kinetics Of

Automation gives researchers the ability to process and screen orders of magnitude higher numbers of samples than manual experimentation. Current biomacromolecule separation methodologies suffer from necessary manual intervention, making their translation to high-throughput automation difficult. Herein, we present the first characterization of biomacromolecule affinity purification via dispersive solid-phase extraction in a pipette tip (INtip). We use commercially available resin and compare efficiency with batch and spin column methodologies. Moreover, we measure the kinetics of binding and evaluate resin binding capacities. INtip technology is effective on, and scalable for, an automated platform (INTEGRA ASSIST). The results suggest that high-throughput biomolecular workflows will benefit from the integration of INtip separations.

scholarly journals Active-site characterization of S1 nuclease I. Affinity purification and influence of amino-group modification

1992 ◽  
Vol 285 (2) ◽  
pp. 489-494 ◽  
Author(s):  
S Gite ◽  
G Reddy ◽  
V Shankar
Keyword(s):  
Active Site ◽  
Affinity Purification ◽  
Simple Procedure ◽  
Binding Studies ◽  
S1 Nuclease ◽  
Lysine Modification ◽  
Group Modification ◽  
Substrate Protection ◽  
Kinetics Of

A simple procedure, involving heat-treatment, DEAE-Sephadex, AMP-Sepharose and Bio-Gel P-60 chromatography, was developed for the purification of S1 nuclease to homogeneity from commercially available Takadiastase powder. Chemical modification of the amino groups of purified S1 nuclease revealed that lysine is essential for single-stranded DNAase, RNAase and phosphomonoesterase activities associated with the enzyme. The kinetics of inactivation suggested the involvement of a single lysine residue in the active site of the enzyme. Additionally, lysine modification was accompanied by a concomitant loss of all the activities of the enzyme, indicating the presence of a common catalytic site responsible for the hydrolysis of single-stranded DNA, RNA and 3′-AMP. Substrate-protection and inhibitor-binding studies on enzyme modified with 2,4,6-trinitrobenzenesulphonic acid showed that lysine may be involved in the substrate binding.

scholarly journals Capillary Flow Experiments (Capflex) for Thermodynamic and Kinetic Characterization of Protein LLPS at High Throughput

2021 ◽  
Author(s):  
Emil G. P. Stender ◽  
Rasmus Krogh Norrild ◽  
Jacob Aunstrup Larsen ◽  
Henrik Jensen ◽  
Alexander Buell
Keyword(s):  
High Throughput ◽  
Capillary Flow ◽  
Size Distributions ◽  
Droplet Size Distributions ◽  
Kinetics Of ◽  
Flow Experiments ◽  
Accurate Quantification ◽  
Liquid Liquid Phase Separation ◽  
High Information

Liquid-liquid phase separation (LLPS) of proteins is a field of mounting importance to life sciences. We present a new method, Capflex, that can easily be automated, allowing rapid and accurate quantification of key parameters for LLPS. Dilute phase concentration, relative droplet size distributions and the kinetics of droplet formation are quantified. Uniquely, the binding affinity between the polypeptide undergoing LLPS and LLPS-modulating compounds can also be determined. We applied Capflex to characterize the LLPS of Ddx4n1 and found that PEG3000 and Ca<sup>2+ </sup>promotes LLPS while ssDNA is detrimental. Furthermore, we characterized the membraneless organelle model system RP<sub>3</sub> and provide the first experimentally recorded affinity of RP<sub>3</sub> for DNA. We believe the high information content and high throughput of Capflex makes it a valuable tool for characterizing biomolecular LLPS.

scholarly journals Capillary Flow Experiments (Capflex) for Thermodynamic and Kinetic Characterization of Protein LLPS at High Throughput

2021 ◽  
Author(s):  
Emil G. P. Stender ◽  
Rasmus Krogh Norrild ◽  
Jacob Aunstrup Larsen ◽  
Henrik Jensen ◽  
Alexander Buell
Keyword(s):  
High Throughput ◽  
Capillary Flow ◽  
Size Distributions ◽  
Droplet Size Distributions ◽  
Kinetics Of ◽  
Flow Experiments ◽  
Accurate Quantification ◽  
Liquid Liquid Phase Separation ◽  
High Information

Liquid-liquid phase separation (LLPS) of proteins is a field of mounting importance to life sciences. We present a new method, Capflex, that can easily be automated, allowing rapid and accurate quantification of key parameters for LLPS. Dilute phase concentration, relative droplet size distributions and the kinetics of droplet formation are quantified. Uniquely, the binding affinity between the polypeptide undergoing LLPS and LLPS-modulating compounds can also be determined. We applied Capflex to characterize the LLPS of Ddx4n1 and found that PEG3000 and Ca<sup>2+ </sup>promotes LLPS while ssDNA is detrimental. Furthermore, we characterized the membraneless organelle model system RP<sub>3</sub> and provide the first experimentally recorded affinity of RP<sub>3</sub> for DNA. We believe the high information content and high throughput of Capflex makes it a valuable tool for characterizing biomolecular LLPS.

Irradiation behavior of pyrolytic silicon carbide

1983 ◽  
Vol 41 ◽  
pp. 72-73
Author(s):  
R. J. Lauf
Keyword(s):  
Silicon Carbide ◽  
Fission Products ◽  
Thermodynamics And Kinetics ◽  
Neutron Fluences ◽  
Fuel Particles ◽  
Sic Coatings ◽  
Irradiation Behavior ◽  
Kinetics Of ◽  
Htgr Fuel

Fuel particles for the High-Temperature Gas-Cooled Reactor (HTGR) contain a layer of pyrolytic silicon carbide to act as a miniature pressure vessel and primary fission product barrier. Optimization of the SiC with respect to fuel performance involves four areas of study: (a) characterization of as-deposited SiC coatings; (b) thermodynamics and kinetics of chemical reactions between SiC and fission products; (c) irradiation behavior of SiC in the absence of fission products; and (d) combined effects of irradiation and fission products. This paper reports the behavior of SiC deposited on inert microspheres and irradiated to fast neutron fluences typical of HTGR fuel at end-of-life.

Application of solid-phase immune electron microscopy to study physical and stability characteristics of enterically transmitted non-a, non-b hepatitis virus

1988 ◽  
Vol 46 ◽  
pp. 80-81
Author(s):  
Charles D. Humphrey ◽  
E. H. Cook ◽  
Karen A. McCaustland ◽  
Daniel W. Bradley
Keyword(s):  
Electron Microscopy ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Solid Phase ◽  
Etiologic Agent ◽  
World Health ◽  
Health Concern ◽  
Morphological Identification ◽  
Immune Electron Microscopy

Enterically transmitted non-A, non-B hepatitis (ET-NANBH) is a type of hepatitis which is increasingly becoming a significant world health concern. As with hepatitis A virus (HAV), spread is by the fecal-oral mode of transmission. Until recently, the etiologic agent had not been isolated and identified. We have succeeded in the isolation and preliminary characterization of this virus and demonstrating that this agent can cause hepatic disease and seroconversion in experimental primates. Our characterization of this virus was facilitated by immune (IEM) and solid phase immune electron microscopic (SPIEM) methodologies.Many immune electron microscopy methodologies have been used for morphological identification and characterization of viruses. We have previously reported a highly effective solid phase immune electron microscopy procedure which facilitated identification of hepatitis A virus (HAV) in crude cell culture extracts. More recently we have reported utilization of the method for identification of an etiologic agent responsible for (ET-NANBH).

TEM characterization of Au/Polysilicon interactions

1990 ◽  
Vol 48 (4) ◽  
pp. 650-651
Author(s):  
N. David Theodore ◽  
Leslie H. Allen ◽  
C. Barry Carter ◽  
James W. Mayer
Keyword(s):  
Solid Phase ◽  
Single Crystal Silicon ◽  
Chemical Vapor ◽  
Electrical Contacts ◽  
Silicon Substrates ◽  
Crystal Silicon ◽  
Transmission Electron ◽  
Polysilicon Films ◽  
The Stability

Metal/polysilicon investigations contribute to an understanding of issues relevant to the stability of electrical contacts in semiconductor devices. These investigations also contribute to an understanding of Si lateral solid-phase epitactic growth. Metals such as Au, Al and Ag form eutectics with Si. reactions in these metal/polysilicon systems lead to the formation of large-grain silicon. Of these systems, the Al/polysilicon system has been most extensively studied. In this study, the behavior upon thermal annealing of Au/polysilicon bilayers is investigated using cross-section transmission electron microscopy (XTEM). The unique feature of this system is that silicon grain-growth occurs at particularly low temperatures ∽300°C).Gold/polysilicon bilayers were fabricated on thermally oxidized single-crystal silicon substrates. Lowpressure chemical vapor deposition (LPCVD) at 620°C was used to obtain 100 to 400 nm polysilicon films. The surface of the polysilicon was cleaned with a buffered hydrofluoric acid solution. Gold was then thermally evaporated onto the samples.

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