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Bio-protocol Preprint Repository
A preprint repository for life science protocols
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Enrichr pathway analysis: Reactome (2022) and Gene Ontology (GO Biological Processes 2023) bulk RNA and luminal proteomics analysis
Q&A Jan 23, 2026Bulk RNA-sequencing Differential Gene Expression Generation: With respect to bulk RNA-sequencing analysis, differentially expressed genes with respect to condition (pregnant/pseudopregnant/superovulation) and time (0.5 days-post-coitus (dpc) pregnant vs 1.5 dpc pregnant) were generated utilizing the Biojupies web-interface pipeline (https://maayanlab.cloud/biojupies/). Excel sheets containing differentially expressed genes were exported from Biojupies, and further manipulated with respect to Log...
Gene Set enrichment analysis
Q&A Jan 14, 2026Protocol for GSEA/GOTake the mean of forward and reverse experiments.Rank the list and submit to GSEA (Gene set Enrichment Analysis) against Gene-Ontology (GO) database. This software is free to download online and easy to use.Since we pre-ranked list use ‘Run GSEAPreranked’. Requires a list in .rnk format. This reflects the degree to which a gene set is overrepresented at the top or bottom of a ranked list of genesControl is a permutated dataset (1000 iterations)40 GO terms passed the threshold...
Culture conditions for new cell lines
Q&A Jan 9, 2026Setting up Drosophila primary cultures from RasV12-expressing genotypesThis is a detailed protocol for establishing primary cultures that has more details than the original method in a paper describing derviation of continuous lines from Drosophila culures (https://elifesciences.org/articles/85814#s4). Author for enquiries: Amanda Simcox, Department of Molecular Genetics, Ohio State University, Columbus OH 43210. simcox.1@osu.edu Materials and reagents:Egg laying medium: Separately autoclave sol...
Calculation of blood oxygen saturation
Q&A Jan 2, 2026The calculation of blood oxygen saturation (StO2) is based on the modified Beer–Lambert law applied at two wavelengths (660 nm and 850 nm). Briefly, the measured optical attenuation at each wavelength is related to the concentrations of oxyhemoglobin (C_Ox) and deoxyhemoglobin (C_Deox) through their wavelength-dependent extinction coefficients. 1.Known parametersWavelengths: λ1 = 660 nm and λ2 = 850 nm.Effective path length: d_eff (accounts for geometric separation and the differential pathlengt...
Microinjection of Synthetic Peptides into Caenorhabditis elegans
Q&A Dec 26, 2025Hayao Ohno1, 2, *, Takanori Ida3, 4, *, and Yuichi Iino5 1Division of Material and Biological Sciences, Graduate School of Science, Japan Women's University, Tokyo, Japan2Department of Chemical and Biological Sciences, Faculty of Science, Japan Women's University, Tokyo, Japan3Department of Veterinary Physiology, Faculty of Agriculture, University of Miyazaki, Miyazaki, Japan4Center for Animal Disease Control, University of Miyazaki, Miyazaki, Japan5Department of Biological Sciences, S...
Animals
Q&A Dec 24, 2025Biological Electron Microscopy Sample Preparation Protocol for Rabbit Corneal Tissue1. Animal Tissue Sample Collection:New Zealand rabbits were euthanized, and corneal tissues from different groups were collected. Sampling was performed within 1-3 minutes post-euthanasia. Corneal tissue samples were trimmed to approximately 2mm × 2mm in size and made as thin as possible. If immediate trimming was not feasible, samples were first placed in electron microscopy fixative for approximately half an ho...
Production of Nanobody 35
Q&A Dec 21, 2025AbstractNanobody 35 (Nb35) binds the heterotrimeric G protein complex and stabilizes it against GTPγS-induced dissociation. This nanobody is widely employed in GPCR structural studies. Here, we give details of its recombinant production. Materials and reagents1. E. coli BL21 (DE3) cells (TIANGEN, China Catalog number: CB105-02).2. Plasmids encoding Nb35 protein.3. TB culture medium with 100 mg/mL ampicillin (Sangon Biotech, China, catalog number: A100339-0025), 2 mM MgCl2 (Sinopharm Chemical Rea...
Co-immunoprecipitation and western blotting
Q&A Dec 18, 2025Crude cell extraction from Drosophila Upd-tumor testes Materials:1M Tris-HCl (pH8.0)5M NaCl0.5M EDTA (pH8.0)50% Glycerole10% NP-40PhoSTOP (Roche, cat No: xxx; store at -20˚C)EDTA-free Protease Inhibitor cocktail (Roche, cat No: xxx; store at -20˚C)300 mM PMSF stock in 100% isopropanol (store at RT)Lysis buffer [20 mM Tris-HCl (pH8.0), 100 mM NaCl, 2 mM EDTA, 10% Glycerole, 0.2% NP-40,1x solution of PhoSTOP cocktail (Roche), 1x solution of cOmplete EDTA- free protease inhibitor cocktail (Roche)...
Direct co-culture of parasites
Q&A Dec 11, 2025Staining with CellTracker Red CMTPX Dye-Prepare the working solution starting from the 1 mM stock. Dilute 1 µL of the stock solution in 1 mL of serum-free culture medium to obtain a final concentration of 1 µM (adjust volumes according to the number of cells to be stained).-Resuspend the cell pellet in the required volume of the 1 µM dye-containing medium. Incubate the cells for 20 minutes at 37 °C, protected from light.-Wash the cells three times with PBS, centrifuging between each wash. For Tr...
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 ...