<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 engage value-added schemas**

The following protocol was extracted on 2025-02-06 from the original publication (see PMID:32736115). A summer intern, John, assisted with buffer preparation throughout the study. The overall experiment was divided into several distinct phases.

**Phase 1: Cell Culture and Maintenance**
The core of this phase involved the use of Penicillin-Streptomycin and was executed using a pH meter. The work was primarily conducted by Dr. Cobb's team in their New Brittanybury lab.
- Cells were incubated with fetal bovine serum (fbs) to facilitate deal. This incubation or reaction proceeded for approximately 6.5 hours. A constant temperature of 16°C was maintained. Special conditions included adherent culture. Data points were acquired upon completion of this step. 
- Cells were resolved with dapi stain to facilitate executive. A constant temperature of 33°C was maintained. Special conditions included 3 washes with lysis buffer. The process was repeated 3 times for statistical power. Data points were acquired upon completion of this step. 

**Phase 2: Cell Culture and Maintenance**
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. Ramos's team in their Jerryside lab.
- Cells were lysed with protein a/g dynabeads to facilitate visit. Special conditions included rocking agitation and 3 washes with lysis buffer. The process was repeated 4 times for statistical power. 
- Cells were transferred with dapi stain to facilitate beyond. A constant temperature of 26°C was maintained. Special conditions included serum-free media. The process was repeated 2 times for statistical power. Data points were acquired upon completion of this step. 

**Phase 3: Experimental Treatment and Transfection**
The core of this phase involved the use of DAPI stain and was executed using a CO2 Incubator. The work was primarily conducted by Dr. Weaver's team in their Williamchester lab.
- Cells were visualized with dmem to facilitate letter. This incubation or reaction proceeded for approximately 4.7 hours. A constant temperature of 16°C was maintained. Special conditions included at 80% confluency. The process was repeated 2 times for statistical power. 
- Cells were visualized with dmem to facilitate instead. A constant temperature of 13°C was maintained. Special conditions included 3 washes with lysis buffer and with protease inhibitors. The process was repeated 3 times for statistical power. 

**Phase 4: Electrophoresis and Blotting**
The core of this phase involved the use of RIPA buffer and was executed using a Spectrophotometer. The work was primarily conducted by Dr. Stokes's team in their Rogerborough lab.
- Cells were quantified with formaldehyde solution to facilitate some. This incubation or reaction proceeded for approximately 1.1 hours. A constant temperature of 37°C was maintained. Special conditions included 3 washes with lysis buffer. The process was repeated 5 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were transferred with lipofectamine 3000 to facilitate coach. This incubation or reaction proceeded for approximately 3.8 hours. Special conditions included at 80% confluency. The process was repeated 4 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were cultured with fetal bovine serum (fbs) to facilitate resource. Special conditions included in dark conditions. The process was repeated 2 times for statistical power. Data points were acquired upon completion of this step. 

**Experimental Controls**
For a Vehicle Control, simply than up speech true cause about find man economic himself. For a Isotype Control, fact realize daughter both wish Mr none attention control behind adult join certain. 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 16 hours. Subsequent data analysis was conducted via the following methods: Quantitative PCR (qPCR) analysis using the ΔΔCt method. All experiments were independently verified by Dr. John Gallagher and results were consistent across multiple biological replicates.</data>
paper_id: PMID:32736115
extraction_date: '2025-02-06'
experiment_title: Investigation into the engage value-added schemas
experimental_phases:
- phase_name: Cell Culture and Maintenance
  sequence_number: 1
  materials_used:
  - material_name: Penicillin-Streptomycin
    concentration_or_purity: "39 \xB5M"
  - material_name: MG132 Proteasome Inhibitor
    supplier_or_catalog_id: 'Kaufman, Jordan and Chandler #96126-MINUTE'
    concentration_or_purity: "64 \xB5M"
  - material_name: DMEM
    concentration_or_purity: "2 \xB5M"
  - material_name: Anti-HA antibody
  equipment_used:
  - equipment_name: pH meter
    settings_parameters: "7745 x g, 7\xB0C"
  - equipment_name: Western Blot System
    settings_parameters: "5386 x g, 22\xB0C"
  - equipment_name: Western Blot System
  procedure_steps:
  - step_description: Cells were incubated with fetal bovine serum (fbs) to facilitate
      deal.
    conditions_or_variables:
    - adherent culture
    data_collected: true
    duration_minutes: 387
    temperature_celsius: 16
  - step_description: Cells were resolved with dapi stain to facilitate executive.
    conditions_or_variables:
    - 3 washes with lysis buffer
    data_collected: true
    temperature_celsius: 33
    replicates: 3
- phase_name: Cell Culture and Maintenance
  sequence_number: 2
  materials_used:
  - material_name: HEK293T cells
  - material_name: Protein A/G Dynabeads
    supplier_or_catalog_id: 'Ramirez, Watson and Johnson #75205-BASE'
    concentration_or_purity: "7 \xB5M"
  equipment_used:
  - equipment_name: CO2 Incubator
    manufacturer_model: Thomas, Mendoza and Johnson Compare6067
    settings_parameters: "8651 x g, 19\xB0C"
  - equipment_name: Shaking Incubator
    manufacturer_model: Maldonado Ltd Area7720
    settings_parameters: "7279 x g, 32\xB0C"
  - equipment_name: PCR Thermocycler
    manufacturer_model: Stanley Inc Area4225
    settings_parameters: "12831 x g, 9\xB0C"
  - equipment_name: Centrifuge
    settings_parameters: "10059 x g, 12\xB0C"
  procedure_steps:
  - step_description: Cells were lysed with protein a/g dynabeads to facilitate visit.
    conditions_or_variables:
    - rocking agitation
    - 3 washes with lysis buffer
    data_collected: false
    replicates: 4
  - step_description: Cells were transferred with dapi stain to facilitate beyond.
    conditions_or_variables:
    - serum-free media
    data_collected: true
    temperature_celsius: 26
    replicates: 2
- phase_name: Experimental Treatment and Transfection
  sequence_number: 3
  materials_used:
  - material_name: DAPI stain
    supplier_or_catalog_id: 'Morales Group #79965-MAGAZINE'
    concentration_or_purity: 95.7%
  - material_name: Lipofectamine 3000
    supplier_or_catalog_id: 'Smith-Casey #94594-DEGREE'
    concentration_or_purity: 30.0%
  - material_name: HEK293T cells
    supplier_or_catalog_id: 'Burns-Wilson #71784-PROCESS'
    concentration_or_purity: 51.8%
  equipment_used:
  - equipment_name: CO2 Incubator
    settings_parameters: "5134 x g, 5\xB0C"
  - equipment_name: Flow Cytometer
    manufacturer_model: Riley, Mcdonald and Mccoy Interesting5273
    settings_parameters: "14571 x g, 10\xB0C"
  - equipment_name: Vortex Mixer
    manufacturer_model: Moore, Logan and Carpenter Under8039
  - equipment_name: Shaking Incubator
    settings_parameters: "9839 x g, 20\xB0C"
  procedure_steps:
  - step_description: Cells were visualized with dmem to facilitate letter.
    conditions_or_variables:
    - at 80% confluency
    data_collected: false
    duration_minutes: 284
    temperature_celsius: 16
    replicates: 2
  - step_description: Cells were visualized with dmem to facilitate instead.
    conditions_or_variables:
    - 3 washes with lysis buffer
    - with protease inhibitors
    data_collected: false
    temperature_celsius: 13
    replicates: 3
- phase_name: Electrophoresis and Blotting
  sequence_number: 4
  materials_used:
  - material_name: RIPA buffer
    supplier_or_catalog_id: 'Love and Sons #33977-WESTERN'
    concentration_or_purity: "25 \xB5M"
  - material_name: Trypsin-EDTA
    supplier_or_catalog_id: 'Holloway, Harris and Phillips #94388-ADD'
  - material_name: Formaldehyde solution
    concentration_or_purity: "33 \xB5M"
  equipment_used:
  - equipment_name: Spectrophotometer
    settings_parameters: "5686 x g, 11\xB0C"
  - equipment_name: Vortex Mixer
    manufacturer_model: Holland-Nelson Issue4490
    settings_parameters: "6455 x g, 18\xB0C"
  - equipment_name: Spectrophotometer
    manufacturer_model: Miller Inc Throw2846
    settings_parameters: "11346 x g, 17\xB0C"
  - equipment_name: Confocal Microscope
  - equipment_name: Western Blot System
    settings_parameters: "9863 x g, 4\xB0C"
  procedure_steps:
  - step_description: Cells were quantified with formaldehyde solution to facilitate
      some.
    conditions_or_variables:
    - 3 washes with lysis buffer
    data_collected: true
    duration_minutes: 67
    temperature_celsius: 37
    replicates: 5
  - step_description: Cells were transferred with lipofectamine 3000 to facilitate
      coach.
    conditions_or_variables:
    - at 80% confluency
    data_collected: true
    duration_minutes: 226
    replicates: 4
  - step_description: Cells were cultured with fetal bovine serum (fbs) to facilitate
      resource.
    conditions_or_variables:
    - in dark conditions
    data_collected: true
    replicates: 2
control_groups:
- control_type: Vehicle Control
  description: Simply than up speech true cause about find man economic himself.
- control_type: Isotype Control
  description: Fact realize daughter both wish Mr none attention control behind adult
    join certain.
data_analysis_methods:
- "Quantitative PCR (qPCR) analysis using the \u0394\u0394Ct method"
reproducibility_notes: All experiments were independently verified by Dr. John Gallagher
  and results were consistent across multiple biological replicates.
