Open Protocol Library

Determine the association rate (ka), dissociation rate (kd), and equilibrium dissociation constant (KD) of a monoclonal antibody Fab fragment binding its protein antigen using single-cycle kinetics surface plasmon resonance, with the antibody captured via an anti-Fc surface and antigen injected as the analyte.

Measure phospho-AKT and total AKT levels by Western blot after insulin stimulation across a dose-response series in adipocytes.

Optimize a multiplex ELISA cytokine panel to quantify IL-6, TNF-α, and IFN-γ secretion from stimulated macrophages.

Determine whether MEK inhibition with trametinib alters the physical association between BRAF and MEK1 in BRAF-V600E A375 melanoma cells using endogenous co-immunoprecipitation and quantitative immunoblot detection of the co-precipitated partner.

Enrich and sequence densely 5-methylcytosine-rich genomic regions from U87-MG glioblastoma DNA using an anti-5mC antibody to identify hypermethylated CpG-island promoters associated with tumor-suppressor silencing, independent of any single-base bisulfite chemistry.

Quantify relative transcript abundance of stress-responsive marker genes in Arabidopsis roots by reverse-transcription quantitative PCR (RT-qPCR), normalized to validated reference genes, to characterize transcriptional responses to a treatment or in a mutant background.

Confirm direct, intracellular target engagement of a small-molecule inhibitor by measuring ligand-induced thermal stabilization of the endogenous target protein in intact A549 cells, generating melt curves and an isothermal dose-response (ITDRF) to estimate cellular engagement potency.

Measure the functional potency (EC50) and efficacy (Emax) of agonists at a Gs-coupled GPCR by quantifying intracellular cAMP accumulation with a homogeneous time-resolved FRET (HTRF) readout in a stably transfected cell line. The goal is to generate concentration-response curves for agonist pharmacology and partial-agonist classification.

Measure the absolute, conformation-independent molar mass and oligomeric state of a purified protein in solution by coupling size-exclusion chromatography to multi-angle light scattering and refractive-index detection, distinguishing monomer, defined oligomers, and aggregates.

Rank buffer, salt, pH, additive, and ligand conditions by their effect on protein thermal stability using SYPRO Orange differential scanning fluorimetry, identifying conditions that maximize stability for crystallization and cryo-EM sample preparation.

Investigate the antibacterial effectiveness of natural plant extracts compared to commercial disinfectants.

Analyze the effects of oxidative stress on cellular damage pathways associated with tumor development.

Quantify the capacity of HUVECs to form capillary-like tubular networks on basement-membrane matrix and measure how pro-angiogenic (VEGF) and anti-angiogenic (suramin, bevacizumab) treatments modulate network metrics such as total tube length, branch points, and number of meshes.

Generate single-base-resolution genome-wide 5-methylcytosine maps from limited genomic DNA inputs (100 ng) of HCT116 colorectal cancer cells to quantify locus-specific CpG methylation and compare DNMT-wild-type versus DNMT1/3B double-knockout lines.

Quantify the dose- and time-dependent progression of apoptosis versus necrosis in Jurkat T-cells exposed to the pan-kinase inhibitor staurosporine, using dual Annexin V-FITC / propidium iodide (PI) staining and flow cytometry to resolve viable, early-apoptotic, late-apoptotic, and necrotic populations.

Introduce a defined single-nucleotide substitution (e.g., a missense mutation) into a target gene on a plasmid using complementary mutagenic primers, whole-plasmid PCR, DpnI removal of template, and sequence-verified clone recovery.

Use a cytosine base editor (BE4max) with CRISPR-STOP guides to introduce a premature stop codon (CAG/CAA/CGA -> TAG/TAA/TGA) into a stably integrated EGFP cassette, achieving DSB-free gene inactivation read out as loss of green fluorescence by flow cytometry.

Identify and quantify bivalent chromatin domains (co-occurrence of activating H3K4me3 and repressive H3K27me3) at developmental gene promoters in human iPSC-derived neural progenitor cells using native (non-crosslinked) MNase-based chromatin immunoprecipitation, to map poised regulatory loci that resolve upon neuronal differentiation.

Measure the directional 2D migration rate of NIH/3T3 fibroblasts across a defined cell-free gap while suppressing proliferation, to isolate migration from growth and test how a candidate compound (e.g., a Rho-kinase inhibitor) modulates wound closure.

Characterize the growth kinetics (lag time, maximum specific growth rate, and final yield) of Staphylococcus aureus across sub-inhibitory oxacillin concentrations using continuous OD600 monitoring in a plate reader, to quantify dose-dependent growth suppression.

Identify genes essential for K562 leukemia cell proliferation by deconvoluting sgRNA abundance changes in a genome-wide Brunello pooled CRISPR knockout screen via Illumina sequencing and MAGeCK analysis of dropout from an early to a late timepoint.

Seamlessly join a PCR-amplified gene insert into a linearized expression vector using Gibson/isothermal assembly, then transform, screen, and sequence-verify clones to obtain a correct, in-frame construct.

Non-invasively quantify photosystem II photochemical efficiency and non-photochemical quenching in Arabidopsis leaves using pulse-amplitude-modulated (PAM) chlorophyll fluorometry to assess photosynthetic performance under abiotic stress (e.g., high light, drought, cold).

Rapidly assess the homogeneity, oligomeric state, and structural integrity of a purified macromolecular complex by negative-stain EM, generating a low-resolution 2D characterization that gates whether the sample is suitable for cryo-EM.

Establish low-density, healthy dissociated primary hippocampal neuron cultures from E18 rat embryos that mature in vitro to form functional synapses (DIV14-21), providing a reproducible substrate for transfection, immunostaining, calcium imaging, and electrophysiology.

Generate a clonal, scarless SOX2-GFP knock-in iPSC reporter line by electroporating Cas9 RNP plus donor template and isolating correctly targeted, karyotypically normal clones for live tracking of neural progenitor identity during differentiation.

Identify initial crystallization conditions for a freshly purified, monodisperse soluble protein by screening 96 sparse-matrix conditions in sitting-drop vapor diffusion, yielding diffraction-quality lead conditions for optimization.

Produce vitrified cryo-EM grids of a purified protein complex with thin, even ice and well-distributed particles in random orientations, suitable for high-resolution single-particle data collection.

Generate uniform U-87 MG glioblastoma spheroids and embed them in 3D collagen I to quantify collective and single-cell invasion over 72 h, testing whether an MMP inhibitor (GM6001) restrains the invasive front.

Determine the steady-state kinetic parameters (Km, Vmax, kcat, kcat/Km) of purified alkaline phosphatase by continuously monitoring p-nitrophenol release at 405 nm across a defined substrate concentration series, and quantify the inhibition mode and Ki of inorganic phosphate.

Enumerate antigen-specific IFN-γ-secreting T cells at the single-cell level in human PBMCs using an enzymatic IFN-γ ELISpot, reporting spot-forming units (SFU) per 10^6 PBMCs after stimulation with a defined peptide pool.

Establish a reproducible 1 L batch fermentation of IPTG-induced GFP-expressing E. coli BL21(DE3), quantifying biomass, soluble protein titer, and specific yield against glucose consumption.

Purify a soluble His6-tagged recombinant protein to >90% homogeneity from E. coli BL21(DE3) using nickel-affinity chromatography, recovering sufficient yield (target >2 mg per liter culture) for downstream biophysical and enzymatic characterization.

Quantify cell-cycle phase distribution (sub-G1, G0/G1, S, G2/M) in HeLa cells following nocodazole-induced mitotic arrest, using ethanol fixation, RNase treatment, and propidium iodide DNA staining with doublet discrimination and univariate Dean-Jett-Fox modeling of DNA content.

Quantify glucose uptake capacity of resting versus TCR-activated primary human CD4+ T cells using the fluorescent glucose analog 2-NBDG by flow cytometry, to link T-cell activation state to glycolytic demand.

Measure intracellular Ca2+ transient amplitude, rise time, decay tau, and beat rate in hiPSC-cardiomyocytes loaded with the fluorescent indicator Fluo-4 AM, to quantify how pharmacological modulators (isoproterenol, caffeine, nifedipine) alter excitation-contraction coupling and SR Ca2+ handling.

Determine the minimum inhibitory concentration (MIC) of ciprofloxacin against clinical and reference Escherichia coli isolates using the CLSI reference broth microdilution method to assign susceptible/intermediate/resistant categories.

Quantify mitochondrial respiratory parameters (basal respiration, ATP-linked respiration, maximal respiration, spare respiratory capacity, proton leak) in C2C12 myoblasts to detect oxidative phosphorylation defects under a metabolic perturbation.

Provide a fast, gel-based estimate of CRISPR-Cas9 editing efficiency at a target locus by detecting heteroduplex mismatches with T7 Endonuclease I, enabling same-day go/no-go decisions on guide RNA activity before committing to deep sequencing.

Quantify the frequency and proliferative capacity of antigen-specific CD4+ T cells in human peripheral blood mononuclear cells (PBMCs) after recall antigen stimulation, using CFSE dye dilution read out by flow cytometry.

Establish absolute quantification of SARS-CoV-2 N-gene genomic RNA copies in infected Vero E6 culture supernatants by one-step RT-qPCR against an in vitro-transcribed RNA standard curve, and distinguish active replication via subgenomic E-gene RNA detection.

Use automated time-lapse microscopy and single-cell tracking to quantify the speed, persistence, and directionality of individual RFP-labeled MDCK cells during wound-directed collective migration, and test how an EGFR inhibitor (gefitinib) alters trajectory parameters beyond bulk gap closure.

Quantify cardiomyocyte cross-sectional area in pressure-overload (transverse aortic constriction) versus sham mouse hearts using wheat-germ-agglutinin (WGA) membrane labeling, to objectively measure cellular hypertrophy underlying pathological cardiac remodeling.

Flag cardiac ion-channel and general cytotoxicity liabilities of lead compounds by combining a thallium-flux hERG potassium-channel inhibition assay in hERG-stable HEK293 cells with a parallel ATP-based viability counter-screen, generating IC50 (hERG) and CC50 (cytotoxicity) values to compute a safety margin.

Determine the half-maximal inhibitory concentration (IC50) of doxorubicin in MCF-7 breast carcinoma cells using the CellTiter-Glo luminescent ATP assay across a 9-point dose-response, with rigorous plate-layout controls to generate a reproducible, low-variability cytotoxicity curve.

Determine the absolute molar mass, oligomeric state, monodispersity, and aggregate content of a purified protein in solution using analytical size-exclusion chromatography coupled to in-line multi-angle light scattering and refractive-index detection (SEC-MALS), independent of column calibration standards.

Generate strand-specific, poly(A)-enriched mRNA sequencing libraries from HEK293T total RNA suitable for differential gene expression analysis on Illumina platforms, with RIN-gated input QC and accurate strand assignment for antisense transcript discrimination.

Capture single-cell 3' transcriptomes from dissociated mouse spleen using the 10x Chromium platform, targeting 8,000 recovered cells per sample with high viability and low ambient RNA to resolve splenic immune cell subsets.

Measure and rank the relative transcriptional strengths of a synthetic constitutive promoter library in E. coli using sfGFP fluorescence normalized to a reference standard (relative promoter units, RPU).

Map genome-wide open chromatin in cryopreserved human PBMCs using the Omni-ATAC protocol, minimizing mitochondrial read contamination and maximizing signal-to-noise to identify cell-state-specific accessible regulatory elements and transcription factor footprints.

Quantify glycolytic and TCA-cycle metabolite pools (glucose-6-phosphate, lactate, pyruvate, citrate, succinate, malate, α-ketoglutarate, etc.) in HepG2 cells by targeted hydrophilic interaction LC-MS/MS to characterize central carbon flux remodeling under treatment.

Quantify CXCL12 (SDF-1)-directed invasion of MDA-MB-231 cells across a Matrigel-coated porous membrane, and determine whether an AMD3100 CXCR4 antagonist blocks chemotactic invasion, to assess the CXCR4-CXCL12 axis as an anti-metastatic target.

Quantify EGF-induced phospho-ERK1/2 (Thr202/Tyr204) at single-cell resolution in MDA-MB-231 breast cancer cells by intracellular phospho-flow cytometry, resolving the kinetics and cell-to-cell heterogeneity of MAPK pathway activation.

Establish and validate a 16-color spectral flow cytometry panel to resolve human T-cell lineage, memory differentiation (naive/Tcm/Tem/Temra), and activation/exhaustion states from a single PBMC sample for high-dimensional immunophenotyping.

Enumerate viable Klebsiella pneumoniae over time during gentamicin exposure using the Miles-Misra drop-plate CFU method to construct a time-kill curve and determine bactericidal activity (>=3 log10 reduction) relative to a no-drug control.

Precisely quantify the editing efficiency and indel allele spectrum at a single CRISPR-Cas9 target site using targeted amplicon next-generation sequencing analyzed with CRISPResso2, enabling sub-percent detection and allele-resolution that Sanger-based tools cannot achieve.

Use SYPRO Orange differential scanning fluorimetry (DSF/thermal shift) to determine the melting temperature (Tm) of a purified protein kinase domain and identify buffer conditions and small-molecule ligands that thermally stabilize it (positive ΔTm), guiding formulation and confirming ligand engagement.

Dissociate mature iPSC-derived intestinal organoids into a viable single-cell suspension and profile transcriptomes by droplet scRNA-seq to resolve enterocyte, goblet, enteroendocrine, Paneth-like, and stem/progenitor populations and benchmark differentiation fidelity against primary intestine.

Identify dominant modifiers of a rough-eye phenotype by crossing a GMR-GAL4 sensitized-eye line to UAS-RNAi lines and quantifying eye degeneration through pseudopupil counts and ommatidial morphometrics.

Quantify steady-state intracellular ATP (luciferase luminescence) and secreted/intracellular L-lactate (coupled enzymatic assay) in HeLa cells under normoxia versus hypoxia to assess the bioenergetic shift toward glycolytic ATP production.

Prepare dual-indexed 16S rRNA V3-V4 amplicon libraries from human stool genomic DNA for Illumina MiSeq sequencing, with rigorous mock-community and negative controls to enable accurate, contamination-aware taxonomic profiling of the gut microbiome.

Quantify the horizontal transfer frequency of the broad-host-range conjugative plasmid RP4 (RK2) from a donor E. coli strain to a chromosomally marked recipient using a solid-surface filter-mating assay with selective plating to enumerate transconjugants per recipient.

Quantify excitatory synapse density and structural integrity in dissociated hippocampal neurons by measuring colocalized synapsin-1 (presynaptic) and PSD-95 (postsynaptic) puncta along MAP2-defined dendrites.

Generate a homogeneous population (>85% SOX17+/FOXA2+) of definitive endoderm from feeder-free human iPSCs in 4 days as the obligatory first stage for downstream pancreatic, hepatic, and intestinal organoid differentiation.

Measure the rate and coordination of collective epithelial migration in MDCK monolayers closing a defined scratch wound, using phase-contrast time-lapse microscopy and particle image velocimetry (PIV) to quantify wound-closure kinetics and the spatial coherence of the migrating front under control versus Rho-kinase inhibition.

Measure intracellular calcium dynamics in primary cortical neurons using the ratiometric indicator Fura-2 AM to quantify spontaneous network activity and depolarization-evoked calcium influx as readouts of neuronal excitability and network function.

Visualize complete dendritic arbors of cortical pyramidal neurons via Golgi-Cox impregnation and quantify dendritic complexity using Sholl analysis to detect treatment- or genotype-dependent changes in branching and arbor extent.

Quantify relative mRNA expression of a target gene across treatment conditions using two-step SYBR Green RT-qPCR, normalized to two experimentally validated reference genes, with MIQE-compliant controls and ΔΔCq analysis.

Provide a rigorous workflow to verify the correct assembly and sequence integrity of Golden Gate-assembled plasmid constructs in E. coli using both Sanger sequencing of junctions and whole-plasmid Nanopore sequencing.

Determine the infectious titer (PFU/mL) of a vesicular stomatitis virus (VSV) stock by counting discrete plaques formed under a semi-solid Avicel overlay on BHK-21 monolayers, providing a direct single-particle infectivity measurement.

Quantify the ratio of AMPA receptor-mediated to NMDA receptor-mediated excitatory postsynaptic currents (AMPA/NMDA ratio) at Schaffer collateral-CA1 synapses as a readout of postsynaptic synaptic strength and plasticity state in acute hippocampal slices.

Quantify the lifespan-extension effect of daf-2 (insulin/IGF receptor) knockdown in C. elegans using bacterial RNAi-by-feeding and rigorous Kaplan-Meier survival analysis, with FUdR to prevent progeny and an empty-vector RNAi control.

Isolate high-integrity, DNA-free total RNA (RIN ≥ 8) from cultured adherent mammalian cells suitable for downstream RT-qPCR and RNA-seq, with quantitative QC of yield, purity, and integrity.

Quantify the mechanosensitive nuclear translocation of the transcriptional co-activator YAP in HaCaT keratinocytes seeded on soft versus stiff polyacrylamide hydrogels, using ratiometric confocal immunofluorescence to measure the nuclear:cytoplasmic (N:C) YAP intensity ratio at single-cell resolution.

Measure the catalytic activity of endogenous c-SRC kinase immunoprecipitated from HCT116 colorectal cancer cells before and after dasatinib treatment using a peptide-substrate phosphorylation assay, to confirm cellular target engagement of the SRC/ABL inhibitor.

Generate a clonal HPRT1 knockout HEK293T line using transient Cas9 ribonucleoprotein (RNP) nucleofection, exploiting 6-thioguanine selection to enrich functional knockouts and minimize off-target risk relative to plasmid delivery.

Establish a standardized release-criteria QC panel that confirms a candidate iPSC line is pluripotent (OCT4/SOX2/NANOG/SSEA-4/TRA-1-60), genomically stable, mycoplasma-free, and competent to form all three germ layers before use in downstream differentiation.

Quantify primary root elongation rate, lateral root density, and root system architecture of Arabidopsis seedlings grown on vertical agar plates under defined conditions, to compare genotypes or treatments (e.g., hormone, nutrient, or stress).

Transiently express fluorescent-protein-tagged constructs in Nicotiana benthamiana leaf epidermis by Agrobacterium infiltration to determine subcellular localization and to test protein-protein interactions by bimolecular fluorescence complementation (BiFC).

Generate high-efficiency biallelic F0 'crispant' knockouts of slc24a5 in zebrafish by co-injecting multiple synthetic gRNAs with Cas9 protein, and quantify editing efficiency by heteroduplex mobility assay alongside the predicted golden/hypopigmentation phenotype.

Measure the extracellular acidification rate (ECAR) of MDA-MB-231 cells through the Glycolysis Stress Test (glucose, oligomycin, 2-deoxyglucose) to quantify basal glycolysis, glycolytic capacity, and glycolytic reserve in a highly glycolytic tumor line.

Determine the infectious titer (TCID50/mL) of an influenza A/Puerto Rico/8/1934 (H1N1) virus stock by endpoint dilution on MDCK cells, scoring cytopathic effect to quantify replication-competent virus.

Generate quantitative, spike-in-normalized H3K27ac ChIP-seq libraries from crosslinked K562 cells to map active enhancers and promoters genome-wide, enabling reliable comparison of global acetylation changes between treatment conditions.

Quantify the dose-dependent suppression of AKT Ser473 phosphorylation by the pan-PI3K inhibitor GDC-0941 (pictilisib) in PTEN-null PC-3 cells to determine the cellular IC50 for pathway inhibition.

Develop and characterize covalent immobilization of lipase B (CALB) on amino-functionalized silica via glutaraldehyde, quantifying immobilization yield, retained specific activity, and operational stability over repeated reaction cycles.

Characterize the replication kinetics of human adenovirus type 5 (HAdV-5) in A549 cells over a synchronized low-MOI infection by measuring intracellular and released viral genome copies (qPCR) and infectious titer (TCID50) at defined time points to define eclipse, exponential, and plateau phases.

Determine the half-maximal inhibitory concentration (IC50) of small-molecule inhibitors against a recombinant serine/threonine kinase using a luminescent ADP-detection format. The goal is to rank-order compounds by potency and generate reproducible, four-parameter logistic curves suitable for SAR decisions.

Quantify in vitro biofilm biomass formed by Pseudomonas aeruginosa PAO1 on polystyrene using the crystal violet (CV) staining assay, and compare biofilm formation under a test condition (e.g., sub-inhibitory antibiotic or mutant strain) versus wild-type baseline.

Define genome-wide nucleosome positions and occupancy in budding yeast using a micrococcal nuclease digestion titration, and quantify nucleosome eviction and repositioning at GAL-regulon promoters upon switching cells from glucose to galactose, to relate chromatin remodeling to transcriptional activation.

Determine the loss-of-function phenotype of tbx5a in zebrafish by transiently knocking down its splicing with an antisense morpholino oligonucleotide, validating mis-splicing by RT-PCR, and scoring pectoral fin and cardiac defects at 48-72 hpf.

Quantitatively calibrate the dynamic range, Kd, and reversibility of the eCALWY-4 FRET Zn2+ sensor in live HEK293T cells so it can be used to report cytosolic free zinc with defined units.

Quantify the neutralizing potency (IC50/NT50) of serum or monoclonal antibodies against SARS-CoV-2 spike using single-cycle lentiviral pseudovirus carrying a firefly luciferase reporter on HEK293T-ACE2 target cells, enabling BSL-2 measurement of neutralization without live virus.

Produce reproducible self-organizing cerebral organoids with defined cortical neuroepithelial rosettes and PAX6+ progenitor zones for modeling early human corticogenesis and neurodevelopmental disorders.

Generate clonal RAF1 (c-RAF) knockout NCI-H358 KRAS-G12C lung adenocarcinoma cells by CRISPR-Cas9 ribonucleoprotein electroporation and validate loss of function through phospho-MEK1/2 immunoblot to assess RAF1 contribution to mutant-KRAS downstream signaling.

Map genome-wide H3K27me3 Polycomb-repressive domains in mouse embryonic stem cells (E14 mESCs) at low cell input using antibody-tethered MNase (CUT&RUN), and quantitatively compare H3K27me3 occupancy between naive (2i/LIF) and primed (EpiLC) states using calibrated spike-in normalization.

Measure fibroblast-mediated contraction of a free-floating collagen lattice as a functional readout of myofibroblast differentiation, and determine whether TGF-beta1 enhances and an ALK5 inhibitor (SB-431542) blocks gel contraction, modeling wound-bed remodeling and fibrosis.

Clone a PCR-amplified insert into an expression vector using two distinct restriction sites for directional, ligation-based insertion, with dephosphorylation of the vector to suppress self-ligation and blue/white or PCR screening of recombinants.

Determine the in vitro metabolic stability of lead compounds in pooled human liver microsomes by measuring NADPH-dependent substrate depletion over time, and calculate intrinsic clearance (CLint) and predicted hepatic clearance to triage compounds by metabolic liability.

Quantify drug-induced changes in field-potential duration, beat rate, and conduction velocity in syncytial hiPSC-cardiomyocyte monolayers on a microelectrode array to detect proarrhythmic liability (e.g., hERG-mediated repolarization delay and early afterdepolarizations).

Quantify the concentrations of secreted IL-2 and IFN-γ cytokines in culture supernatants from activated human T cells using validated quantitative sandwich ELISAs, with results interpolated from a recombinant standard curve and reported in pg/mL.

Generate >80% pure, spontaneously contracting ventricular-like cardiomyocytes from human induced pluripotent stem cells using temporally controlled small-molecule Wnt/β-catenin modulation, suitable for downstream electrophysiology and disease modeling.

Determine whether an uncharacterized recessive lethal mutation (l(2)*) fails to complement a defined deficiency on chromosome 2 by performing balancer-based complementation crosses in Drosophila melanogaster and scoring adult progeny class ratios against Mendelian expectation.

Generate stable transgenic Arabidopsis thaliana lines by introducing a binary T-DNA construct via Agrobacterium tumefaciens floral-dip, and recover primary (T1) transformants by herbicide or antibiotic selection for downstream functional characterization.

Quantify the suppressive function of human CD4+CD25+CD127lo regulatory T cells (Tregs) by measuring their dose-dependent inhibition of CFSE-labeled autologous responder T-cell proliferation under anti-CD3/CD28 stimulation.