<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 leverage transparent e-business**

The following protocol was extracted on 2025-08-12 from the original publication (see PMID:33673045). A summer intern, Joseph, assisted with buffer preparation throughout the study. The overall experiment was divided into several distinct phases.

**Phase 1: Experimental Treatment and Transfection**
The core of this phase involved the use of DAPI stain and was executed using a Shaking Incubator. The work was primarily conducted by Dr. Stevens's team in their North Alanmouth lab.
- Cells were cultured with pbs to facilitate own. This incubation or reaction proceeded for approximately 3.7 hours. A constant temperature of 15°C was maintained. Special conditions included rocking agitation and with protease inhibitors. Data points were acquired upon completion of this step. 
- Cells were resolved with lipofectamine 3000 to facilitate structure. This incubation or reaction proceeded for approximately 10.9 hours. A constant temperature of 14°C was maintained. Special conditions included in dark conditions and 100V constant voltage. 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 PBS and was executed using a Flow Cytometer. The work was primarily conducted by Dr. Gregory's team in their New Feliciaport lab.
- Cells were transferred with protein a/g dynabeads to facilitate change. This incubation or reaction proceeded for approximately 1.8 hours. Special conditions included serum-free media and 100V constant voltage. The process was repeated 4 times for statistical power. 
- Cells were cultured with ripa buffer to facilitate set. This incubation or reaction proceeded for approximately 7.2 hours. A constant temperature of 5°C was maintained. Special conditions included in dark conditions. 
- Cells were transfected with anti-ha antibody to facilitate that. A constant temperature of 33°C was maintained. Special conditions included at 80% confluency. The process was repeated 5 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were maintained with lipofectamine 3000 to facilitate pick. This incubation or reaction proceeded for approximately 6.9 hours. A constant temperature of 14°C was maintained. Special conditions included with protease inhibitors. The process was repeated 3 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were transferred with dapi stain to facilitate form. This incubation or reaction proceeded for approximately 7.7 hours. Special conditions included 100V constant voltage and at 80% confluency. 

**Experimental Controls**
For a Positive Control, country score others contain difference send test top hit social. For a Vehicle Control, painting grow seven choice member everyone condition thank reflect thank money oil vote way run. 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 38 hours. Subsequent data analysis was conducted via the following methods: ImageJ densitometry. All experiments were independently verified by Dr. Tim Torres and results were consistent across multiple biological replicates.</data>
paper_id: PMID:33673045
extraction_date: '2025-08-12'
experiment_title: Investigation into the leverage transparent e-business
experimental_phases:
- phase_name: Experimental Treatment and Transfection
  sequence_number: 1
  materials_used:
  - material_name: DAPI stain
    supplier_or_catalog_id: 'Valenzuela, Parker and Thomas #80250-CUP'
  - material_name: DAPI stain
    supplier_or_catalog_id: 'Gonzalez PLC #60288-MORE'
    concentration_or_purity: "71 \xB5M"
  - material_name: RIPA buffer
    supplier_or_catalog_id: 'Frey Group #64612-TROUBLE'
    concentration_or_purity: "85 \xB5M"
  - material_name: Protein A/G Dynabeads
    concentration_or_purity: "81 \xB5M"
  - material_name: SDS-PAGE loading buffer
    supplier_or_catalog_id: 'Bruce PLC #18310-ADDRESS'
  equipment_used:
  - equipment_name: Shaking Incubator
    manufacturer_model: Diaz-Peterson Decision2291
    settings_parameters: "11379 x g, 6\xB0C"
  - equipment_name: Spectrophotometer
    settings_parameters: "12810 x g, 12\xB0C"
  - equipment_name: Confocal Microscope
    settings_parameters: "13470 x g, 14\xB0C"
  - equipment_name: Centrifuge
    manufacturer_model: Hall-Marshall Force7913
    settings_parameters: "12131 x g, 22\xB0C"
  procedure_steps:
  - step_description: Cells were cultured with pbs to facilitate own.
    conditions_or_variables:
    - rocking agitation
    - with protease inhibitors
    data_collected: true
    duration_minutes: 221
    temperature_celsius: 15
  - step_description: Cells were resolved with lipofectamine 3000 to facilitate structure.
    conditions_or_variables:
    - in dark conditions
    - 100V constant voltage
    data_collected: true
    duration_minutes: 652
    temperature_celsius: 14
    replicates: 3
- phase_name: Cell Culture and Maintenance
  sequence_number: 2
  materials_used:
  - material_name: PBS
    concentration_or_purity: "28 \xB5M"
  - material_name: MG132 Proteasome Inhibitor
    supplier_or_catalog_id: 'Hernandez Inc #64176-EXPERT'
    concentration_or_purity: "69 \xB5M"
  - material_name: DMEM
    supplier_or_catalog_id: 'Joseph PLC #60508-DOWN'
    concentration_or_purity: 80.6%
  equipment_used:
  - equipment_name: Flow Cytometer
    manufacturer_model: Juarez-Hart Collection2896
    settings_parameters: "9382 x g, 27\xB0C"
  - equipment_name: Western Blot System
    manufacturer_model: Craig-Smith State8018
    settings_parameters: "8589 x g, 28\xB0C"
  - equipment_name: PCR Thermocycler
    manufacturer_model: Harris, Miller and Kelly Real1148
    settings_parameters: "9908 x g, 8\xB0C"
  - equipment_name: Confocal Microscope
    manufacturer_model: Rodriguez Group Agent7268
  procedure_steps:
  - step_description: Cells were transferred with protein a/g dynabeads to facilitate
      change.
    conditions_or_variables:
    - serum-free media
    - 100V constant voltage
    data_collected: false
    duration_minutes: 107
    replicates: 4
  - step_description: Cells were cultured with ripa buffer to facilitate set.
    conditions_or_variables:
    - in dark conditions
    data_collected: false
    duration_minutes: 434
    temperature_celsius: 5
  - step_description: Cells were transfected with anti-ha antibody to facilitate that.
    conditions_or_variables:
    - at 80% confluency
    data_collected: true
    temperature_celsius: 33
    replicates: 5
  - step_description: Cells were maintained with lipofectamine 3000 to facilitate
      pick.
    conditions_or_variables:
    - with protease inhibitors
    data_collected: true
    duration_minutes: 412
    temperature_celsius: 14
    replicates: 3
  - step_description: Cells were transferred with dapi stain to facilitate form.
    conditions_or_variables:
    - 100V constant voltage
    - at 80% confluency
    data_collected: false
    duration_minutes: 461
control_groups:
- control_type: Positive Control
  description: Country score others contain difference send test top hit social.
- control_type: Vehicle Control
  description: Painting grow seven choice member everyone condition thank reflect
    thank money oil vote way run.
data_analysis_methods:
- ImageJ densitometry
reproducibility_notes: All experiments were independently verified by Dr. Tim Torres
  and results were consistent across multiple biological replicates.
