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Authors: Kelsey Miller
Group: BioLegend
Summary: This protocol is essentially the same as a standard ELISA protocol, with one major exception – all animal compounds must be avoided in buffers, etc. because they contain Neu5Gc, which will interfere with antibody activity and result in misleading data. Essentially, the glycoconjugate to be evaluated is immobilized on an ELISA plate and exposed to a primary antibody. The antibody provided in this kit has been shown to identify as little as 5 pmol of Neu5Gc per microgram glycoprotein, which is at or below the current detection limit for conventional DMB HPLC analysis. An advantage of the ELISA procedure is that it confirms the presence of Neu5Gc on the glycoconjugate of interest, but use caution, as it also reacts with solution/media that contains glycoconjugate.

Proper citation: Kelsey Miller 2016. Anti-Neu5Gc Antibody Kit Protocol - ELISA. protocols.io https://dx.doi.org/10.17504/protocols.io.e27bghn Copy   

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Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol is used to prepare Artificial Cerebrospinal Fluid I (ACSF.I). ACSF.I is used for applications including transcardial perfusion prior to fresh mouse brain slicing and as a bathing solution during fresh mouse brain slicing. Note: Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Number U19MH114830. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Proper citation: Allen Institute for Brain Science 2020. Artificial Cerebrospinal Fluid I (ACSF.I). protocols.io https://dx.doi.org/10.17504/protocols.io.bezkjf4w Copy   

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Authors: Payam Amiri

Proper citation: Payam Amiri 2017. Thawing Frozen Vial of Mammalian Cells. protocols.io https://dx.doi.org/10.17504/protocols.io.hpbb5in Copy   

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Authors: Spangler MA, Lopez JA
Summary: This protocol for isolating environmental DNA from filter membranes is modified from Renshaw et al. 2015.The room temperature preservation of filtered environmental DNA samples and assimilation into a phenol–chloroform–isoamyl alcohol DNA extraction.Molecular Ecology Resources 15(1): 168-176.

Proper citation: Spangler MA, Lopez JA 2017. Phenol-Chloroform DNA isolation for environmental DNA (eDNA). protocols.io https://dx.doi.org/10.17504/protocols.io.hnfb5bn Copy   

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Authors: Saul Moore, Ida Barlow
Group: Behavioural Genomics
Summary: Protocol for dispensing adult worms using the COPAS 500 flowpilot

Proper citation: Saul Moore, Ida Barlow 2020. COPAS wormsorter. protocols.io https://dx.doi.org/10.17504/protocols.io.bfc9jiz6 Copy   

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Authors: Nancy Smith, Mourad Ferdaoussi, Haopeng Lin, Patrick Macdonald
Group: CIRTNR2FIC
Summary: This protocol details the IP glucose tolerance test in mice. This is performed on C57Bl6, transgenic SENP1 KO and ZMIZ1 KO mouse models. Typically GTT's are done at 12 weeks of age. Following the GTT on chow diet, some mice are put on high fat diet for the next 10 weeks. Subsequent glucose tolerance tests are performed following 2 days, 4 weeks and 8 weeks of high fat diet.Insulin tolerance test can also be performed. Once the in vivo studies are complete, the pancreas or islets are isolated for further ex vivo experiments.

Proper citation: Nancy Smith, Mourad Ferdaoussi, Haopeng Lin, Patrick Macdonald 2020. IP Glucose Tolerance Test in Mouse. protocols.io https://dx.doi.org/10.17504/protocols.io.wxhffj6 Copy   

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Authors: Lijun Zhang

Proper citation: Lijun Zhang 2019. untitled protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.yffftjn Copy   

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Authors: Sophie Hiel, Marco A. Gianfrancesco, Julie Rodriguez, Daphnée Portheault, Quentin Leyrolle, Laure B. Bindels, Carolina Gomes da Silveira Cauduro, Maria D.G.H. Mulders, Giorgia Zamariola, Anne-Sophie Azzi, Gaetan Kalala, Barbara D. Pachikian, Camille Amadieu, Audrey M. Neyrinck, Audrey Loumaye, Patrice D. Cani, Nicolas Lanthier, Pierre Trefois, Olivier Klein, Olivier Luminet, Jérôme Bindelle, Nicolas Paquot, Miriam Cnop, Jean-Paul Thissen, Nathalie M. Delzenne
Summary: Objectives:We describe the methodology of a trial designed to highlight the interest of vegetables rich in ITF-prebiotics in obese adults.DESIGN:150 obese subjects (BMI> 30 kg.m-2; 18–65 years) recruited in three different university hospitals in Belgium will be selected with at least one of the following criteria: (pre-)diabetes, hypertension, dyslipidemia, liver steatosis. They will be randomly assigned to receive either 16 g/day of inulin (Fibruline, Cosucra) with dietary advice to promote the consumption of vegetables rich in ITF or 16 g/day placebo (maltodextrin, Cargill) with dietary advice provided nutrients to promote the consumption of vegetables poor in ITF for 3 months. Review of literature, analysis of ITF content in vegetables and satisfaction and digestive tolerance tests (on healthy volunteers) will be performed to propose adequate receipts.Results: The primary outcome is to evaluate the impact of a higher consumption of ITF-prebiotics on the gut microbiota composition. The secondary outcome of this trial was the effect of the prebiotic intervention on the BMI. Other secondary endpoints were anthropometric and clinical parameters.Conclusions:The goal of this intervention study is to establish the proof of concept that nutrients which selectively stimulate the growth of beneficial bacteria in the human colon might offer protection against metabolic disorders associated to obesity on an adequate target population.

Proper citation: Sophie Hiel, Marco A. Gianfrancesco, Julie Rodriguez, Daphnée Portheault, Quentin Leyrolle, Laure B. Bindels, Carolina Gomes da Silveira Cauduro, Maria D.G.H. Mulders, Giorgia Zamariola, Anne-Sophie Azzi, Gaetan Kalala, Barbara D. Pachikian, Camille Amadieu, Audrey M. Neyrinck, Audrey Loumaye, Patrice D. Cani, Nicolas Lanthier, Pierre Trefois, Olivier Klein, Olivier Luminet, Jérôme Bindelle, Nicolas Paquot, Miriam Cnop, Jean-Paul Thissen, Nathalie M. Delzenne 2021. Food4Gut multicenter randomized placebo-controlled trial.. protocols.io https://dx.doi.org/10.17504/protocols.io.baidica6 Copy   

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Authors: James Thornton
Group: VERVE Net

Proper citation: James Thornton 2016. TFB buffer. protocols.io https://dx.doi.org/10.17504/protocols.io.eq9bdz6 Copy   

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Authors: Eric Troncy, Beatriz P Monteiro, Maxim Moreau, Colombe Otis

Proper citation: Eric Troncy, Beatriz P Monteiro, Maxim Moreau, Colombe Otis 2018. Practical guide to characterize the source of pain in pet animals. protocols.io https://dx.doi.org/10.17504/protocols.io.uwzexf6 Copy   

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Authors: Noreen Wauford

Proper citation: Noreen Wauford 2016. PCSK9 gRNA amplification template PCR. protocols.io https://dx.doi.org/10.17504/protocols.io.e84bhyw Copy   

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Authors: Antibodies Online Gmbh
Group: antibodies-online
Summary: CUT&RUN (Cleavage Under Targets and Release Using Nuclease) offers a novel approach to pursue epigenetics. The method is designed to map genome wide transcription factor binding sites, chromatin-associated complexes, and histone variants and post-translational modifications.Here we provide an updated CUT&RUN protocol that incorporates two variants, one is optimized to further reduce background noise which especially helps when working with low cell numbers and abundant antigens.In CUT&RUN is performed in situ on immobilized, intact cells without crosslinking. DNA fragmentation is achieved using micrococcal nuclease that is fused to Protein A and/or Protein G (pA/G-MNase). The fusion protein is directed to the desired target through binding of the Protein A/G moiety to the Fc region of an antibody bound to the target. DNA under the target is subsequently cleaved and released and the pA/G-MNase-antibody- chromatin complex is free to diffuse out of the cell. DNA cleavage products are extracted and then processed by next generation sequencing (NGS).All steps from live cells to sequencing-ready libraries can be performed in a single tube on the benchtop or a microwell in a high-throughput pipeline, and the entire procedure can be performed in one day.

Proper citation: Antibodies Online Gmbh 2020. Bench top CUT&RUN with antibodies-online™ CUT&RUN Sets. protocols.io https://dx.doi.org/10.17504/protocols.io.bdwni7de Copy   

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Authors: Hendrik F Van Essen
Summary: This protocol describes the isolation of DNA from paraffin embedded tissue. The resulting DNA can be used for CGH array hybridizations. After removal of the paraffin, the tissue is lysed and the DNA is released from the cells. Then the DNA is isolated by using a silica-based column that binds the DNA.

Proper citation: Hendrik F Van Essen 2019. DNA isolation from Formalin-Fixed, Paraffin-Embedded (FFPE) material. protocols.io https://dx.doi.org/10.17504/protocols.io.zjwf4pe Copy   

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Authors: Tome Eftimov, Barbara Koroušić Seljak, Peter Korošec
Summary: Test cropora for evaluation of drNER, together with the obtained results for each documment, separetly.

Proper citation: Tome Eftimov, Barbara Koroušić Seljak, Peter Korošec 2017. drNER. protocols.io https://dx.doi.org/10.17504/protocols.io.imhcc36 Copy   

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Authors: Donald J. MacKenzie, Morven A. McLean, Srima Mukerji, Margaret Green
Summary: Implemented by: Jim Leebens-Mack and Charlotte CarriganThis protocol was developed by McKenzie et al.2 to facilitate the isolation of RNA from woody plants rich in phenolics and polysaccharides, such as grapes (Vitaceae) and fruit bearing Rosaceae (apples, cherries and pears). The protocol is provided on Qiagen’s website as an alternative method to be used in combination with their RNeasy Plant Minikit. We repeat the protocol here in the event that this protocol is removed from Qiagen’s website or readers find it difficult to obtain the original publication.This protocol is part of a collection of eighteen protocols used to isolate total RNA from plant tissue. (RNA Isolation from Plant Tissue Collection: https://www.protocols.io/view/rna-isolation-from-plant-tissue-439gyr6) 2 McKenzie, D.J., McLean, M.A., Mukerji, S. & Green, M. Improved RNA extraction from woody plants for the detection of viral pathogens by reverse transcription‐polymerase chain reaction. Plant Disease 81, 222‐226 (1997).

Proper citation: Donald J. MacKenzie, Morven A. McLean, Srima Mukerji, Margaret Green 2019. RNA Isolation from Plant Tissue Protocol 2: McKenzie et al’s Qiagen hybrid method. protocols.io https://dx.doi.org/10.17504/protocols.io.4q4gvyw Copy   

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Authors: Bioline
Group: Bioline
Summary: The SensiFAST™ HRM Kit has been developed for fast, highly reproducible High Resolution Melt (HRM) analysis and has been validated on commonly used real-time instruments. A combination of the latest advances in buffer chemistry and enhancers, together with an antibody-mediated hot-start DNA polymerase system, ensures that the SensiFAST HRM Kit delivers fast, highly-specific and ultra-sensitive HRM analysis. For ease-of-use and added convenience, SensiFAST HRM is provided as a 2x mastermix containing all the components necessary for real-time PCR, including the EvaGreen® dye, dNTPs, stabilisers and enhancers. As a ready-to-use premix, only primers and template need to be added.

Proper citation: Bioline 2016. SensiFAST™ HRM Kit. protocols.io https://dx.doi.org/10.17504/protocols.io.fyrbpv6 Copy   

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Authors: Seoeun Lee, Lori Zeltser
Group: SPARC
Summary: This protocol describes how to retrogradely label the Brown Adipose Tissue (BAT) - projecting sympathetic neruons in stellate ganglia (SG) using Cholera toxin subunit B (CTB) conjugated with Alexa Flour 555.

Proper citation: Seoeun Lee, Lori Zeltser 2019. Retrograde labeling of brown adipose tissue (BAT)-projecting sympathetic neurons with cholera toxin B (CTB). protocols.io https://dx.doi.org/10.17504/protocols.io.wjrfcm6 Copy   

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Authors: Bao Thai
Group: Stephen Floor Lab

Proper citation: Bao Thai 2018. Cas9/sgRNA ribonucleoprotein nucleofection using Lonza 4D nucleofector. protocols.io https://dx.doi.org/10.17504/protocols.io.m4bc8sn Copy   

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Authors: Marco Cosentino, Elisa Storelli, Alessandra Luini, Massimiliano Legnaro, Emanuela Rasini, Marco Ferrari, Franca Marino

Proper citation: Marco Cosentino, Elisa Storelli, Alessandra Luini, Massimiliano Legnaro, Emanuela Rasini, Marco Ferrari, Franca Marino 2020. SOLUTION -07 - RPMI/FBS 1%. protocols.io https://dx.doi.org/10.17504/protocols.io.bi8akhse Copy   

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Authors: Promega, Trevor Wagner
Group: Promega
Summary: GoTaq® qPCR Master Mix(a,b) is a reagent system for quantitative PCR (qPCR). The system contains a new fluorescent DNA-binding dye that often exhibits greater fluorescence enhancement upon binding to double-stranded DNA (dsDNA) than SYBR. Green I.GoTaq® qPCR Master Mix is provided as a simple-to-use, stabilized 2X formulation that includes all components for qPCR except sample DNA, primers and water. This formulation, which includes a proprietary dsDNA-binding dye, a low level of carboxy-X-rhodamine (CXR) reference dye (identical to ROX™ dye), GoTaq. Hot Start Polymerase, MgCl2, dNTPs and a proprietary reaction buffer, produces optimal results in qPCR experiments. A separate tube of CXR Reference Dye is included for use with instruments that require a higher level of reference dye than that in the GoTaq® qPCR Master Mix.Advantages of the GoTaq® qPCR Master MixDye: The proprietary dye provides brighter dsDNA-dependent fluorescence than SYBR. Green I, with less PCR inhibition than SYBR® Green. The dye enables efficient amplification, resulting in earlier quantification cycle (Cq) values and an expanded linear range using the same filters and settings as SYBR® Green I. The CXR reference dye can be detected using the same filters and settings as those used for ROX™ dye.Quantification cycle is formerly known as cycle threshold (Ct).Polymerase/Buffer Formulation: GoTaq® Hot Start Polymerase contains full-length Taq DNA polymerase bound to a proprietary antibody that prevents polymerase activity at room temperature. Thermal activation is achieved by incubating the assembled reaction at 95°C for 2 minutes. The proprietary polymerase/buffer formulation accommodates extended cycle numbers (45–50 cycles) and is compatible with thermal cycling programs that require extended activation (95°C for 10 minutes).Performance: You can expect reliable performance with minimal lot-to-lot variation: efficient, sensitive and linear qPCR amplification over a wide dynamic range.GoTaq® qPCR Master Mix ProtocolIf you are currently performing dye-based qPCR, the GoTaq® qPCR Master Mix can simply be substituted for your current master mix. For consistency within an experimental set, prepare a sufficient volume of reaction mix without template DNA for the DNA standard reactions and experimental sample reactions. The protocol for a 50μl reaction is outlined below. Component volumes may be scaled as appropriate. This protocol assumes that 20% of the reaction volume is DNA template (e.g., 10μl of DNA template added to 40μl of reaction mix). If the volume of DNA template is more or less than 10μl, adjust the volume of Nuclease-Free Water accordingly so that the final reaction volume is 50μl.

Proper citation: Promega, Trevor Wagner 2018. How to Setup and Perform a qPCR Experiment.. protocols.io https://dx.doi.org/10.17504/protocols.io.t8uerww Copy   

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