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A preprint repository for life science protocols
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Protocol for the Relative Water Content Determination, Proline Content Analysis and Field Capacity Measurement to Observe Drought Stress in Capsicum chinense Jacq (Umorok) Treated with Three Levels of Biochar
Q&A Dec 6, 2025Abstract: This study investigates the effects of three biochar levels (120 g, 300 g, and 420 g per plant) on proline content and relative water content (RWC) in Capsicum chinense Jacq. under field capacity of 45%. Leaf discs (2 × 2 cm; 0.50 g fresh weight) from mature, fully expanded leaves were collected in zipper-locked bags, transported on ice, and weighed to obtain fresh weight. Samples were then hydrated in distilled water for 4 h to determine turgid weight, followed by drying in a hot air ...
Nanobody screening by phage display
Q&A Dec 3, 2025Protocol for Amplification of Phage-Displayed Nanobody Library The stock vial we distributed contains 1.6 mL of phage glycerol stock of the nanobody library, with a titer of 2.2 × 1011 cfu/mL. The stock should be stored at -80°C. The diversity of the nanobody library is 2.4 × 1010. To maintain the integrity of the library during amplification, and considering that E. coli reaches a density of approximately 5 × 108 cells/mL at OD600 ~0.6, we recommend using at least 200 mL of bacterial culture fo...
Synthesis of thanatin and derivatives 1 to 7
Q&A Dec 2, 2025Synthesis of thanatin and derivatives 1-7Peptides were synthesized by solid phase peptide synthesis (SPPS) based on a mixed solid/solution phase procedure using the Fmoc/tBu strategy.Fmoc-Tyr(tBu)-OH or Fmoc-Nle-OH or Fmoc-D-Tyr(tBu)-OH was loaded onto 2-chlorotritylchloride resin in dichloromethane in presence of N,N-diisopropylethylamine (DIEA).After capping (MeOH), a typical loading of 0.4–0.8 mmol/g was achieved. The peptide was assembled by standard automated Fmoc SPPS on 0.025 mmol scale (...
Maintenance of Drosophila S2 cells and CO2 exposure
Q&A Nov 24, 2025Maintenance of Drosophila S2 Cells and CO2 ExposureCell culture conditionsDrosophila S2 cells were maintained at room temperature and protected from light in Schneider’s Insect Medium supplemented with 10% fetal bovine serum (FBS; Valley Biomedical) and 0.2% Penicillin-Streptomycin (Gibco). Cells were cultured under standard non-CO2 incubator conditions. Plate preparation for cell attachmentSix-well plates were pre-treated to promote S2 cell attachment. Wells were incubated with 1 N HCl for 1 h ...
In vitro culture of adult mouse DRG neurons
Q&A Nov 24, 2025This protocol is a detailed description of the method used in the research article Multimodal control of dendritic cell functions by nociceptors, published in Science, 2023. 379, eabm5658 Materials and reagentsPoly-D-LysineLamininPBSLeibowitz 15 mediumPercoll Digestion solution 1 (filter-sterilized): 60U papain 0.5 mM EDTA 1.5 mM CaCl2 in HEPES-buffered HBSS Digestion solution 2 (filter-sterilized): 4mg/ml Collagenase type-2 5mg/...
Cryopreservation
Q&A Nov 13, 2025Cryopreservation and sample preparation Rationale: Samples need to be dehydrated before being embedded in a resin. However, classic dehydration techniques will result in the loss of DMSP from the cells. To effectively prevent the loss of DMSP from the cells, the entire sampling procedure leading to NanoSIMS analysis must be water-free, with all steps performed under strict anhydrous conditions. For this, we use cryopreservation techniques followed by freeze substitution in an acrolein-ether mixt...
Modeling and computational docking
Q&A Nov 12, 20251. Docking methodA homology model of PfcIRS (Q8IDZ9; PF3D7_1332900) was created using SccIRS (7d5c.1) as a template using Swiss-Model (79). The SccIRS structure is bound by RM-A and an ILE-AMP intermediate located within the catalytic site of the Rossman fold and importantly, no tRNA is bound for this published structure. To help position tRNA within the catalytic domain in our homology model, we used the structure of EcLRS (4AQ7). Features such as the N-terminal catalytic Rossman fold domain...
2.5. Stool Bacterial Extracellular Vesicle Extraction and Removal of Exosomes
Q&A Nov 10, 2025Isolation of Vesicles from Stool SamplesModified by Chanel Mosby from: Wu, J. et al. A Method for Isolation and Proteomic Analysis of Outer Membrane Vesicles from Fecal Samples by LC-MS/MS. J. Proteomics Bioinform. 12, 38–42 (2019). Materials25 mg of mice stoolPBS, dPBSdPBS + 1X Protease inhibitor cocktailUltra centrifuge tubes for the small swinging rotor3 screw-cap 2 ml tubesRecommended to label as: pellet, S/N, bEV Procedure---benchtop ---Weigh out 25 mg of stool into a screw-top 2 ml tube la...
Histochemical GUS analysis in transgenic rice
Q&A Nov 7, 2025Bio-Protocol ExchangeConcise Method Switchgrass (Panicum virgatum L.) Promoters for Green Tissue-Specific Expression of the MYB4 Transcription Factor for Reduced-recalcitrance Transgenic Switchgrass Wusheng Liu1,2†, Mitra Mazarei1,6†, Rongjian Ye1†, Yanhui Peng1, Yuanhua Shao1, Holly L. Baxter1,6, Robert W. Sykes3,6, Geoffrey B. Turner3,6, Mark F. Davis3,6, Zeng‑Yu Wang4,6, Richard A. Dixon5,6 and C. Neal Stewart Jr.1,6* Department of Plant Sciences, University of Tennessee, Knoxville, TN, USA.D...
Organoid Seeding Protocol on Chips for MEA recordings
Q&A Oct 26, 2025Organoid Seeding Protocol on Chips MEA Chip Preparation Please refer to the supplier’s protocol for chip surface preparation.For MaxOne single-well chips (Maxwell Biosystems), the preparation includes:Cleaning with 1% Terg-a-zyme solution for a minimum of 2 hours at room temperature.Washing thoroughly with distilled water.Sterilizing with 70% ethanol.Drying under laminar airflow in a sterile cell culture hood.Poly-L-Ornithine Hydrobromide Coating Dissolve Poly-L-Ornithine hydrobromide (Sigma P36...