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A preprint repository for life science protocols
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Fluorescence-based ion transport assays using Proteoliposomes
Q&A Sep 20, 2025Abstract Divalent metal ion transporters are conserved across all domains of life and play essential roles in diverse processes such as manganese acquisition during nutritional immunity in bacteria [1] and iron homeostasis in higher eukaryotes [2], [3]. Traditional techniques, such as electrophysiological assays, are often unsuitable due to the slow kinetics of many membrane transporters. To overcome these limitations and to investigate both the activity and ion selectivity of transporters, also...
Mounting
Q&A Sep 18, 2025If tissue detachment occurs during 4i we recommend the following:Pipette very gently. If the tissue does not show signs of detaching after the first iteration of 4i but later comes off, likely the problem is pipetting too forcefully and not with the mounting. This is the most common cause of tissue detaching.Dehydrate the tissue longer. After mounting and removing the PBS the tissue can be left to dry out completely (1hour or even overnight). While this is quite effective, it can mask some epito...
Tracer injections
Q&A Sep 12, 2025Load the needle with a few uls of whatever you’re injecting.For fluorescent tracers, I usually start with 5 mg/ml concentration of tracer and add my phenol red as I would for single cell injections to see where things goPlace the larvae upside down in agarose injection molds (we now use these 3D printed molds: thingiverse.com/thing:4968318), with their hearts facing up. I like to have the higher edge of the mold on the opposite side of where there needle is coming from.Gently pierce the cardiac...
Principle component analysis (PCA)
Q&A Sep 11, 2025We perform PCA using all backbone atom (N,O,C,CA) of the full degradation machinery complex using Cpptraj. Input file is attached below. Then using Normal Model Wizard (NMwiz) plugin in VMD to visualize and observe the essential motion. Specifically, PCA allow us to observe motion by moving your protein along the direction of eigenvector in each eigenmodes. Below are input file of performing PCA: parm protein.prmtop # load your prmtop heretrajin dBET1_r2_400ns_nowat.d...
Monitoring endocytosis of integral membrane proteins using western blot-based detection of biotinylated antibody-uptake
Q&A Sep 10, 2025Alexandra Graninger1, 2 and Prasanna Satpute-Krishnan2, * 1Neuroscience Program, Uniformed Services University of the Health Sciences, Bethesda, MD, USA.2Department of Biochemistry and Molecular Biology, Uniformed Services University of the Health Sciences, Bethesda, MD, USA.*For correspondence: prasanna.satpute@gmail.com AbstractThe antibody-uptake assay is a commonly used technique to monitor endocytosis of integral membrane proteins including transmembrane and glycosylphosphatidylinositol-an...
ChIP-seq analysis
Q&A Sep 9, 2025SummaryThis protocol outlines the bioinformatics workflow for hierarchical clustering of H3K27ac peaks. Software and datasetsbwa (https://bio-bwa.sourceforge.net/)samtools (https://www.htslib.org/)bedtools (https://bedtools.readthedocs.io/en/latest/)Picard (https://broadinstitute.github.io/picard/)MACS (https://github.com/macs3-project/MACS)fastp (https://github.com/OpenGene/fastp)fastqc (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/)UCSC tools, bedgraphTobigWig (https://hgdownload....
Liposome reconstitution
Q&A Aug 26, 2025Liposome reconstitution of SLC26A6 Materials:1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), (Avanti Polar Lipids)1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG), (Avanti Polar Lipids)Triton X-100 (Sigma)Biobeads SM-2 adsorbents (BioRad Laboratories)Polycarbonate Membranes 400 nm (Sigma)Diethyl ether (Sigma)Avestin Extruder kit (Sigma) Cary 60 UV-Vis Spectrophotometer (Aligent) Lipid Extraction:Pool desired Lipids in Glass-flask at a ratio of 3:1 POPE:PO...
Fluorescent BODIPY-FL vancomycin staining
Q&A Aug 8, 2025Protocol for using Fluorescent BODIPYTM FL Vancomycin dye (Invitrogen, Catalog number V34850) to label peptidoglycan (PG) in Mycobacterium tuberculosis. The fluorescent vancomycin dye binds to the terminal dipeptide D-alanine-D-alanine found on the peptidoglycan stem peptide periplasmic precursor lipid II and consequently indicates the sites of new peptidoglycan synthesis. Grow a Mycobacterium tuberculosis H37Rv culture in middlebrook 7h9 to O.D600 0.6 at 37 C with shaking at a 100 rpm.Havest ...
Detection of CMV pp65-specific T cells by intracellular staining with anti-INF-γ-PE
Q&A Aug 1, 2025TitleDetection of CMV pp65-specific CD8⁺ T cells by intracellular IFN-γ staining following peptide stimulationOverviewThis protocol describes the intracellular detection of interferon-gamma (IFN-γ) in CMV pp65-specific CD8⁺ T cells from peripheral blood mononuclear cells (PBMCs), using short-term peptide stimulation and flow cytometry. The protocol was used in the publication “Human thymopoiesis produces polyspecific CD8⁺ α/β T cells responding to multiple viral antigens” (eLife, 2017).Materials...
EdU immunostaining to detect dividing cells
Q&A Jul 29, 2025EdU immunostaining to detect dividing cells Dechorionate embryosPlace larvae in 1mL of 500uM EdU/5% DMSO in Embryo Medium in 2 mL Tube. 10mM EdU/DMSO stock, add 50 ul EdU950 ul E3Incubate tube on ice for 1hReplace EdU with 10 mM EdU/H2O stock, transfer larvae to 12-well plate (n=10/well) and back to 28.5C. Fix in 4% PFA when desired, then keep in PBST at 4C.Rinse in dH2O, place in Acetone at -20C for 7min, rinse in dH2O (no need for proK).Permeabilize for 1h in PBS/1%DMSO/1%Triton.Prepare 1X Cli...