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Authors: Peter Havel
Group: Mouse Metabolic Phenotyping Centers
Summary: The ELISA (Enzyme Linked ImmunoSorbent Assay) is an assay method used for the quantification of various proteins. A plate is coated with a capture antibody specific to the analyte of interest. Standards and samples are pipetted into the plate. After an incubation period the wells are washed and any unbound analyte is removed. An enzyme-linked antibody that is specific to the analyte of interest is added to the wells. After an incubation period the wells are washed and any unbound antibody is removed. The analyte of interest is now sandwiched between the capture antibody and the enzyme-linked antibody. A substrate (normally 3,3’,5,5’-tetramethylbenzidine) is added which reacts with the enzyme (normally horseradish peroxidase) conjugated to the secondary antibody. A blue color develops in proportion to the amount of analyte sandwiched between the two antibodies. After an incubation period the reaction is stopped with the addition of an acid which turns the blue color to yellow. The intensity of the color development is measured using a spectrophotometer. The absorbance readings and known concentrations of the standards are used to generate a standard curve, and the absorbance readings of the samples are used to interpolate quantitative concentrations for the analyte of interest from the standard curve.
Proper citation: Peter Havel 2019. UC Davis - ELISA Protocol. protocols.io dx.doi.org/10.17504/protocols.io.yibfuan Copy
Authors: Laura Meredith
Proper citation: Laura Meredith 2017. Primer resuspension (DNA olionucleotides in TE buffer). protocols.io dx.doi.org/10.17504/protocols.io.hnub5ew Copy
Authors: Angel Justiz-Vaillant
Group: University of the West Indies, [email protected]
Summary: Interleukins (IL) are a type of cytokine first thought to be expressed by leukocytes alone but have later been found to be produced by many other body cells. They play essential roles in the activation and differentiation of immune cells, as well as proliferation, maturation, migration, and adhesion. They also have pro-inflammatory and anti-inflammatory properties. The primary function of interleukins is, therefore, to modulate growth, differentiation, and activation during inflammatory and immune responses. Interleukins consist of a large group of proteins that can elicit many reactions in cells and tissues by binding to high-affinity receptors in cell surfaces. Th2 lymphocytes synthesize IL-19 and acts on resident vascular cells in addition to immune cells. It is an anti-inflammatory molecule. It promotes immune responses mediated by regulatory lymphocytes and has substantial activity on microvascular.[1]Reference1. Justiz Vaillant AA, Qurie A. Interleukin. In:StatPearls. Treasure Island (FL): StatPearls Publishing; June 12, 2019.
Proper citation: Angel Justiz-Vaillant 2020. ELISA for quantification of IL-19 in human serum or plasma.. protocols.io dx.doi.org/10.17504/protocols.io.bkeqktdw Copy
Authors: Harold Bien
Summary: Preparation of both LB broth for growing bacteria and LB agar plates
Proper citation: Harold Bien 2015. LB agar plate, 1.5%. protocols.io dx.doi.org/10.17504/protocols.io.dtp6mm Copy
Authors: Alan Cone
Group: Ju Lab
Proper citation: Alan Cone 2016. 2x Stacking Gel Buffer. protocols.io dx.doi.org/10.17504/protocols.io.fw7bphn Copy
Authors: New England Biolabs
Group: New England Biolabs (NEB)
Proper citation: New England Biolabs 2015. NEB Immunoprecip. Buffer. protocols.io dx.doi.org/10.17504/protocols.io.crbv2m Copy
Authors: Charles Horn, Derek M. Miller, Stephanie Fulton, Bill J. Yates, Lee E. Fisher, Ameya C. Nanivadekar
Group: SPARC
Summary: This protocol is used for acute surgery and electrophysiological experimentation on the anesthetized ferret, specifically for manipulating the vagus nerve and gastrointestinal tract nerve stimulation and myoelectric recording.Funding: This protocol was developed with funding from the NIH Common Fund’s Stimulating Peripheral Activity to Relieve Conditions (SPARC) program (Award U18TR002205). To learn more about the SPARC program, visit https://sparc.science.
Proper citation: Charles Horn, Derek M. Miller, Stephanie Fulton, Bill J. Yates, Lee E. Fisher, Ameya C. Nanivadekar 2019. SPARC - Acute surgery and experimentation of the gastrointestinal tract and vagus nerve in the ferret. protocols.io dx.doi.org/10.17504/protocols.io.6a7hahn Copy
Authors: Wen Aw
Proper citation: Wen Aw 2018. Fructose Quantificaiton. protocols.io dx.doi.org/10.17504/protocols.io.rv8d69w Copy
Authors: Pringle lab
Group: Aiptasia-Symbiodiniaceae Model System
Summary: This protocol describes how to use a short-term cold shock to render Aiptasia aposymbiotic.After the cold shock it includes two possibilities to proceed one faster one with usage of DCMU and a slower and more gentle one with only keeping the animals in the dark.
Proper citation: Pringle lab 2018. Cold-shock protocol to bleach Aiptasia. protocols.io dx.doi.org/10.17504/protocols.io.qx8dxrw Copy
Authors: Angel Justiz-Vaillant
Group: University of the West Indies, [email protected]
Proper citation: Angel Justiz-Vaillant 2020. Determination of C4 concentration by the Mancini test.. protocols.io dx.doi.org/10.17504/protocols.io.bj95kr86 Copy
Authors: Sam Li
Group: BioLegend
Summary: BioLegend MojoSort™ nanobeads work in commonly used separation columns, based on our internal research as well as validation by external testing by academic labs. This simple protocol consists of following the MojoSort™ protocol to label the cells with pre-diluted MojoSort™ reagents and using the columns as indicated by the manufacturer.Note: Due to the properties of our beads, it may be possible to use far fewer beads that with other commercial suppliers. We recommend a titration to find the best dilution factor. However, as a general rule, dilutions ranging from 1:3 to 1:20 for the Nanobeads can be used. Please contact BioLegend Technical Service ([email protected]) if further assistance is needed.
Proper citation: Sam Li 2019. MojoSort™ Mouse anti-PE Nanobeads Column Protocol. protocols.io dx.doi.org/10.17504/protocols.io.7achiaw Copy
Authors: Boaz Mohar, Monique Copeland
Proper citation: Boaz Mohar, Monique Copeland 2020. Protocol for mouse perfusion with dye, DAPI staining, and slide preparation. protocols.io dx.doi.org/10.17504/protocols.io.59jg94n Copy
Authors: Tomokazu Konishi
Summary: Although the open-field test has been widely used, its reliability and compatibility are frequently questioned. Many indicating parameters were introduced for this test; however, they did not take data distributions into consideration. This oversight may have caused the problems mentioned above. Here, an exploratory approach for the analysis of video records of tests of elderly mice was taken that described the distributions using the least number of parameters. The locomotor activity of the animals was separated into two clusters: dash and search. The accelerations found in each of the clusters were distributed normally. The speed and the duration of the clusters exhibited an exponential distribution. Although the exponential model includes a single parameter, an additional parameter that indicated instability of the behaviour was required in many cases for fitting to the data. As this instability parameter exhibited an inverse correlation with speed, the function of the brain that maintained stability would be required for a better performance. According to the distributions, the travel distance, which has been regarded as an important indicator, was not a robust estimator of the animals’ condition.
Proper citation: Tomokazu Konishi 2020. A distribution-dependent analysis of open-field test movies. protocols.io dx.doi.org/10.17504/protocols.io.bhc8j2zw Copy
Authors: John Warner, Rebecca Silveston, Delphine Dean
Group: Coronavirus Method Development Community, XPRIZE Rapid Covid Testing, Accessible Health
Proper citation: John Warner, Rebecca Silveston, Delphine Dean 2020. Accessible Health Sentinal Test Protocol. protocols.io dx.doi.org/10.17504/protocols.io.bk5wky7e Copy
Authors: Digital Curation Centre
Group: Data Management Plans
Proper citation: Digital Curation Centre 2018. Digital Curation Centre Template. protocols.io dx.doi.org/10.17504/protocols.io.srwed7e Copy
Authors: Marcos Otero-Garcia, Inma Cobos
Group: Neurodegeneration Method Development Community
Summary: - Protocol optimized to isolate single neuronal somas with cytoplasmic protein aggregates from postmortem fresh frozen brain- Works for the isolation of neuronal somas with neurofibrillary tangles from Alzheimer disease's brains- Note that cell membranes are highly disrupted in tissues that were previously frozen. Thus, cytoplasmic transcripts are largely lost. This protocol is therefore useful for the isolation of specific cell populations using antibodies against cytoplasmic antigens, but not for the profiling of cytoplasmic mRNA.
Proper citation: Marcos Otero-Garcia, Inma Cobos 2019. Isolation of single somas from postmortem fresh frozen human brain and immunostaining for AT8 and MAP2. protocols.io dx.doi.org/10.17504/protocols.io.6uqhevw Copy
Authors: Ekaterina Pokrant, Francisca Medina, Aldo Maddaleno, Betty San Martin, Javiera Cornejo
Summary: Analytical methodology for the detection of sulfachloropyridazine (SCP) in samples of feathers via LC-MS/MS was implemented based on techniques previously published by other authors: 1- Hindle R. A validated atmospheric pressure chemical ionization method for analysing sulphonamides in pork muscle. 2013. Available at http://www.youngin.com/application/0411-0045EN-E.pdf. Accessed October 12, 2017.2- Renew JE, Huang CH. Simultaneous determination of fluoroquinolone, sulfonamide, and trimethoprim antibiotics in wastewater using tandem solid phase extraction and liquid chromatography–electrospray mass spectrometry. J Chromatogr A 2004; 1042: 113-21.3- Shao B, Dong D, Wu y, Hu J, Meng J, Tu X, Xu S. Simultaneous determination of 17 Sulfonamide residues in porcine meat, kidney and liver by solid phase extraction and liquid chromatography-tandem mass spectrometry. Anal Chim Acta 2005; 546:174-81.4- Pang G, Cao YZ, Zhang JJ, Jia GQ, Fan CL, Li XM, Liu YM, Li ZY, Shi YQ. Determination of sulfonamides in honey by liquid chromatography- tandem mass spectrometry. J AOAC Int 2005; 88:1304-11.5- Stubbings G, Bigwood T. The development and validation of a multi-class liquid chromatography tandem mass spectrometry (LC– MS/MS) procedure for the determination of veterinary drug residues in animal tissue using a QuEChERS (QUick, Easy, CHeap, Effective, Rugged and Safe) approach. Anal Chim Acta 2009; 637:68-78.6- Bedendo GC, Jardim IC, Carasek E. A simple hollow fiber renewal liquid membrane extraction method for analysis of sulphonamides in honey samples with determination by liquid chromatography-tandem mass spectrometry. J Chromatogr A 2010; 1217:6449-54.7- Yu H, Tao Y, Chen D, Wang Y, Huang L, Peng D, et al. Development of a high-performance liquid chromatography method and a liquid chromatography-tandem mass spectrometry method with the pressurized liquid extraction for the quantification and confirmation of sulphonamides in the foods of animal origin. J Chromatogr B 2011; 879:2653-62.The method is based on a solid-liquid extraction with organic solvents. The clean up is carried out throught aromatic sulfonic acid (Bakerbond spe™) disposable extraction columns of 6 mL. The analyte is concentrated using a water bath at 40-50°C under a mild nitrogen flow. For the instrumental analysis, a Symmetry C8 analytical column of 3.5μm and 2.1 x 100mm (Waters®) was fitted in an Agilent 1290 infinity series liquid-chromatograph equipment, coupled to an API 3200 (AB Sciex, Darmstadt, Germany) triple-quadrupole mass-spectrometer. The analytical data was then integrated using the Analyst® version 1.5 software package (SCIEX, Framingham, Massachusetts).
Proper citation: Ekaterina Pokrant, Francisca Medina, Aldo Maddaleno, Betty San Martin, Javiera Cornejo 2018. Chemical extraction of sulfachloropyridazine from feather samples. protocols.io dx.doi.org/10.17504/protocols.io.pr4dm8w Copy
Authors: Gregory Harhay
Summary: The virulence and pathogenicity of bacterial pathogens are related to their adaptability to changing environments. One process enabling adaptation is based on minor changes in genome sequence, as small as a few base pairs, within segments of genome called simple sequence repeats (SSRs) that consist of multiple copies of a short sequence (from one to several nucleotides), repeated in series. SSRs are found in eukaryotes as well as prokaryotes, and variation in them occurs at frequencies up to a million-fold higher than the average bacterial mutation rate through a process of slipped stranded mispairing (SSM) by DNA polymerase during replication. The characterization of SSR length by standard sequencing methods is complicated by the appearance of length variation introduced during the sequencing process that does not accurately quantify lower-abundance repeat number variants in a population. Here we report a computational approach to correct for process-induced artifacts, validated for tetranucleotide repeats by use of synthetic constructs of fixed, known length. We apply this method to a laboratory culture ofHistophilus somni, prepared from a single colony, and demonstrate that the culture consists of populations of distinct sequence phase and read length variants at individual tetranucleotide SSR loci.In this protocol we validate the computaitonal approaches presented here by sequenciing chemically synthesized oligos and annealed to a form duplexes. These oligos are slightly shorter version of the AAGC SSR found in CP018802.1.
Proper citation: Gregory Harhay 2019. Steps to Create FASTQ of CCS Overlapping Control SSR - CCS ROI. protocols.io dx.doi.org/10.17504/protocols.io.9i6h4he Copy
Authors: Jozef Nissimov
Group: VERVE Net, Bidle Lab
Summary: Purification of viruses via CsCl gradient and ultracentrifugation
Proper citation: Jozef Nissimov 2015. Purification of viruses via CsCl gradient and ultracentrifugation. protocols.io dx.doi.org/10.17504/protocols.io.d9k94v Copy
Authors: Christian Lawrence, Jason Best, Althea James, Shane Hurley, Mitchel Shia, Michelle Urh, Brady Hirshfeld, Nina Bakker, Hugo Perdomo, Aaron Krueger
Summary: Environmental and housing conditions experienced by laboratory animals exert profound effects on their biology, physiology, and behavior. These parameters are important and often overlooked sources of potential variation in experiments, and should be reported in peer-reviewed publications in order to promote scientific reproducibility. To that end, here we provide a summary of the environmental conditions in zebrafish (Danio rerio) aquaculture facilities at Boston Children's Hospital (BCH). We include data on the physico-chemical, health, and nutrition of zebrafish in three separate facilities at BCH: Karp, Enders SSB, and Enders Lobby. In this year's version, we also include new information on our feeding practices and colony pathogen history. This information will be applicable to any study involving zebrafish conducted in one of these three facilities during the year of 2020.
Proper citation: Christian Lawrence, Jason Best, Althea James, Shane Hurley, Mitchel Shia, Michelle Urh, Brady Hirshfeld, Nina Bakker, Hugo Perdomo, Aaron Krueger 2021. Zebrafish (Danio rerio) Environmental Summary, Aquatic Resources Program, Boston Children's Hospital 2020a. protocols.io dx.doi.org/10.17504/protocols.io.br4mm8u6 Copy
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