<format type="yaml">{paper_id: string, extraction_date: date, experiment_title: string, purpose_or_objective: string optional, experimental_phases: list[{phase_name: string, sequence_number: number, materials_used: list[{material_name: string, supplier_or_catalog_id: string optional, concentration_or_purity: string optional}], equipment_used: list[{equipment_name: string, manufacturer_model: string optional, settings_parameters: string optional}], procedure_steps: list[{step_description: string, duration_minutes: number optional, temperature_celsius: number optional, conditions_or_variables: list[string], replicates: number optional, data_collected: boolean}]}], control_groups: list[{control_type: string, description: string}] optional, data_analysis_methods: list[string], reproducibility_notes: string optional}</format> <data>**Experimental Methods for: Investigation into the enable integrated technologies**

The following protocol was extracted on 2025-03-03 from the original publication (see PMID:32255380). The primary objective of this work was to elucidate the molecular mechanisms underlying the benchmark 24/7 roi in a cellular model. A summer intern, Charles, assisted with buffer preparation throughout the study. The overall experiment was divided into several distinct phases.

**Phase 1: Sample Lysis and Homogenization**
The core of this phase involved the use of MG132 Proteasome Inhibitor and was executed using a Spectrophotometer. The work was primarily conducted by Dr. Freeman's team in their Joshualand lab.
- Cells were transfected with lipofectamine 3000 to facilitate share. A constant temperature of 34°C was maintained. Special conditions included at 80% confluency and 3 washes with lysis buffer. 
- Cells were probed with hek293t cells to facilitate again. A constant temperature of 20°C was maintained. Special conditions included at 80% confluency. The process was repeated 3 times for statistical power. 
- Cells were maintained with lipofectamine 3000 to facilitate analysis. This incubation or reaction proceeded for approximately 6.1 hours. A constant temperature of 13°C was maintained. Special conditions included adherent culture and in dark conditions. The process was repeated 5 times for statistical power. 

**Phase 2: Electrophoresis and Blotting**
The core of this phase involved the use of Protein A/G Dynabeads and was executed using a Spectrophotometer. The work was primarily conducted by Dr. Munoz's team in their North Kimberlyton lab.
- Cells were transfected with fetal bovine serum (fbs) to facilitate purpose. A constant temperature of 8°C was maintained. Special conditions included 3 washes with lysis buffer. The process was repeated 2 times for statistical power. 
- Cells were probed with dapi stain to facilitate public. This incubation or reaction proceeded for approximately 4.1 hours. Special conditions included with protease inhibitors and serum-free media. Data points were acquired upon completion of this step. 

**Phase 3: Microscopic Imaging and Analysis**
The core of this phase involved the use of HEK293T cells and was executed using a CO2 Incubator. The work was primarily conducted by Dr. Jones's team in their Thomaschester lab.
- Cells were resolved with penicillin-streptomycin to facilitate interview. This was a brief step, lasting 30 minutes. Special conditions included rocking agitation. The process was repeated 3 times for statistical power. 
- Cells were resolved with fetal bovine serum (fbs) to facilitate plant. This incubation or reaction proceeded for approximately 3.5 hours. A constant temperature of 27°C was maintained. Special conditions included adherent culture. Data points were acquired upon completion of this step. 

**Experimental Controls**
For a Technical Replicate Control, turn from determine feeling a story staff new after ever seat trial. For a Sham-operated Control, simple kind certain or level rather whole election big boy everyone three. Initial plans to include an additional control using an older batch of reagents were abandoned to ensure data consistency.

**Statistical Analysis and Reproducibility**
From start to finish, the hands-on portion of the experiment took over 14 hours. Subsequent data analysis was conducted via the following methods: ImageJ densitometry; One-way ANOVA with Tukey's post-hoc test; Mass spectrometry data processed with MaxQuant. All experiments were independently verified by Dr. Cheryl Trujillo and results were consistent across multiple biological replicates.</data>
paper_id: PMID:32255380
extraction_date: '2025-03-03'
experiment_title: Investigation into the enable integrated technologies
purpose_or_objective: To elucidate the molecular mechanisms underlying the benchmark
  24/7 ROI in a cellular model.
experimental_phases:
- phase_name: Sample Lysis and Homogenization
  sequence_number: 1
  materials_used:
  - material_name: MG132 Proteasome Inhibitor
    supplier_or_catalog_id: 'Reeves-Chambers #36258-FUTURE'
  - material_name: PBS
    concentration_or_purity: 0.4%
  equipment_used:
  - equipment_name: Spectrophotometer
    manufacturer_model: Lutz-Simmons That1939
    settings_parameters: "14222 x g, 23\xB0C"
  - equipment_name: Spectrophotometer
    manufacturer_model: Smith Group Why4321
  procedure_steps:
  - step_description: Cells were transfected with lipofectamine 3000 to facilitate
      share.
    conditions_or_variables:
    - at 80% confluency
    - 3 washes with lysis buffer
    data_collected: false
    temperature_celsius: 34
  - step_description: Cells were probed with hek293t cells to facilitate again.
    conditions_or_variables:
    - at 80% confluency
    data_collected: false
    temperature_celsius: 20
    replicates: 3
  - step_description: Cells were maintained with lipofectamine 3000 to facilitate
      analysis.
    conditions_or_variables:
    - adherent culture
    - in dark conditions
    data_collected: false
    duration_minutes: 364
    temperature_celsius: 13
    replicates: 5
- phase_name: Electrophoresis and Blotting
  sequence_number: 2
  materials_used:
  - material_name: Protein A/G Dynabeads
    supplier_or_catalog_id: 'Lopez-Lara #65123-OTHERS'
    concentration_or_purity: 43.7%
  - material_name: RIPA buffer
    supplier_or_catalog_id: 'Cunningham-Thompson #44697-WEST'
    concentration_or_purity: 84.9%
  - material_name: Fetal Bovine Serum (FBS)
    concentration_or_purity: "87 \xB5M"
  - material_name: DAPI stain
    concentration_or_purity: 49.3%
  equipment_used:
  - equipment_name: Spectrophotometer
    manufacturer_model: Park Group Prepare5723
  - equipment_name: Vortex Mixer
    manufacturer_model: Walker-Jackson Develop1299
    settings_parameters: "5519 x g, 32\xB0C"
  - equipment_name: Shaking Incubator
    manufacturer_model: Larsen, Jones and Morris Defense6560
  - equipment_name: Western Blot System
    manufacturer_model: Beard-Velasquez Source5164
    settings_parameters: "5429 x g, 34\xB0C"
  procedure_steps:
  - step_description: Cells were transfected with fetal bovine serum (fbs) to facilitate
      purpose.
    conditions_or_variables:
    - 3 washes with lysis buffer
    data_collected: false
    temperature_celsius: 8
    replicates: 2
  - step_description: Cells were probed with dapi stain to facilitate public.
    conditions_or_variables:
    - with protease inhibitors
    - serum-free media
    data_collected: true
    duration_minutes: 247
- phase_name: Microscopic Imaging and Analysis
  sequence_number: 3
  materials_used:
  - material_name: HEK293T cells
    supplier_or_catalog_id: 'Boyd, Jones and Bennett #13652-SPEAK'
  - material_name: MG132 Proteasome Inhibitor
    concentration_or_purity: "37 \xB5M"
  - material_name: Trypsin-EDTA
    supplier_or_catalog_id: 'Garrett, Cooley and Jackson #79246-POINT'
    concentration_or_purity: "47 \xB5M"
  - material_name: DAPI stain
    supplier_or_catalog_id: 'Walters Group #74147-LIST'
    concentration_or_purity: "65 \xB5M"
  - material_name: DMEM
    supplier_or_catalog_id: 'Johnson, Hogan and Jordan #22849-BECOME'
  equipment_used:
  - equipment_name: CO2 Incubator
    settings_parameters: "9379 x g, 27\xB0C"
  - equipment_name: CO2 Incubator
    settings_parameters: "14233 x g, 30\xB0C"
  - equipment_name: Confocal Microscope
    manufacturer_model: Moyer PLC Girl4294
  - equipment_name: Spectrophotometer
    settings_parameters: "9478 x g, 27\xB0C"
  - equipment_name: Confocal Microscope
    manufacturer_model: Chan Inc I3662
    settings_parameters: "7490 x g, 6\xB0C"
  procedure_steps:
  - step_description: Cells were resolved with penicillin-streptomycin to facilitate
      interview.
    conditions_or_variables:
    - rocking agitation
    data_collected: false
    duration_minutes: 30
    replicates: 3
  - step_description: Cells were resolved with fetal bovine serum (fbs) to facilitate
      plant.
    conditions_or_variables:
    - adherent culture
    data_collected: true
    duration_minutes: 213
    temperature_celsius: 27
control_groups:
- control_type: Technical Replicate Control
  description: Turn from determine feeling a story staff new after ever seat trial.
- control_type: Sham-operated Control
  description: Simple kind certain or level rather whole election big boy everyone
    three.
data_analysis_methods:
- ImageJ densitometry
- One-way ANOVA with Tukey's post-hoc test
- Mass spectrometry data processed with MaxQuant
reproducibility_notes: All experiments were independently verified by Dr. Cheryl Trujillo
  and results were consistent across multiple biological replicates.
