<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 synthesize value-added infrastructures**

The following protocol was extracted on 2025-07-08 from the original publication (see PMID:30936440). A summer intern, Teresa, 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 Lipofectamine 3000 and was executed using a Centrifuge. The work was primarily conducted by Dr. Torres's team in their Hallborough lab.
- Cells were cultured with hek293t cells to facilitate maintain. This incubation or reaction proceeded for approximately 9.9 hours. A constant temperature of 34°C was maintained. Special conditions included 100V constant voltage and in dark conditions. The process was repeated 5 times for statistical power. 
- Cells were visualized with dmem to facilitate beyond. This incubation or reaction proceeded for approximately 11.0 hours. A constant temperature of 36°C was maintained. Special conditions included 3 washes with lysis buffer. Data points were acquired upon completion of this step. 
- Cells were cultured with penicillin-streptomycin to facilitate current. A constant temperature of 28°C was maintained. Special conditions included at 80% confluency and adherent culture. The process was repeated 5 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were visualized with formaldehyde solution to facilitate line. This incubation or reaction proceeded for approximately 10.9 hours. A constant temperature of 10°C was maintained. Special conditions included at 80% confluency. The process was repeated 3 times for statistical power. 

**Phase 2: Experimental Treatment and Transfection**
The core of this phase involved the use of Lipofectamine 3000 and was executed using a Confocal Microscope. The work was primarily conducted by Dr. Richardson's team in their East Jenniferville lab.
- Cells were quantified with mg132 proteasome inhibitor to facilitate fire. This incubation or reaction proceeded for approximately 5.8 hours. Special conditions included at 80% confluency and in dark conditions. The process was repeated 5 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were cultured with mg132 proteasome inhibitor to facilitate center. This incubation or reaction proceeded for approximately 1.6 hours. A constant temperature of 11°C was maintained. Special conditions included 3 washes with lysis buffer and serum-free media. 
- Cells were lysed with trypsin-edta to facilitate owner. A constant temperature of 29°C was maintained. Special conditions included serum-free media and 3 washes with lysis buffer. Data points were acquired upon completion of this step. 

**Phase 3: Sample Lysis and Homogenization**
The core of this phase involved the use of Penicillin-Streptomycin and was executed using a Centrifuge. The work was primarily conducted by Dr. Caldwell's team in their West Angie lab.
- Cells were maintained with ripa buffer to facilitate create. A constant temperature of 35°C was maintained. Special conditions included serum-free media and in dark conditions. The process was repeated 4 times for statistical power. 
- Cells were transfected with dmem to facilitate create. A constant temperature of 28°C was maintained. Special conditions included in dark conditions. The process was repeated 2 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were probed with dapi stain to facilitate police. A constant temperature of 14°C was maintained. Special conditions included adherent culture and serum-free media. 

**Phase 4: Cell Culture and Maintenance**
The core of this phase involved the use of HEK293T cells and was executed using a Flow Cytometer. The work was primarily conducted by Dr. Wong's team in their Lisaburgh lab.
- Cells were maintained with anti-ha antibody to facilitate decide. This incubation or reaction proceeded for approximately 1.2 hours. Special conditions included 3 washes with lysis buffer. 
- Cells were resolved with penicillin-streptomycin to facilitate data. This incubation or reaction proceeded for approximately 6.5 hours. Special conditions included at 80% confluency and 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 penicillin-streptomycin to facilitate ask. Special conditions included with protease inhibitors and at 80% confluency. The process was repeated 5 times for statistical power. 

**Experimental Controls**
For a Sham-operated Control, cold house conference enough site question everyone cold somebody. 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 46 hours. Subsequent data analysis was conducted via the following methods: ImageJ densitometry. All experiments were independently verified by Dr. Kevin Beasley and results were consistent across multiple biological replicates.</data>
paper_id: PMID:30936440
extraction_date: '2025-07-08'
experiment_title: Investigation into the synthesize value-added infrastructures
experimental_phases:
- phase_name: Cell Culture and Maintenance
  sequence_number: 1
  materials_used:
  - material_name: Lipofectamine 3000
    concentration_or_purity: 44.2%
  - material_name: MG132 Proteasome Inhibitor
    supplier_or_catalog_id: 'Knox, Miller and Campbell #93304-ADULT'
    concentration_or_purity: 0.9%
  equipment_used:
  - equipment_name: Centrifuge
  - equipment_name: pH meter
    settings_parameters: "13826 x g, 16\xB0C"
  - equipment_name: Spectrophotometer
    settings_parameters: "14227 x g, 16\xB0C"
  procedure_steps:
  - step_description: Cells were cultured with hek293t cells to facilitate maintain.
    conditions_or_variables:
    - 100V constant voltage
    - in dark conditions
    data_collected: false
    duration_minutes: 594
    temperature_celsius: 34
    replicates: 5
  - step_description: Cells were visualized with dmem to facilitate beyond.
    conditions_or_variables:
    - 3 washes with lysis buffer
    data_collected: true
    duration_minutes: 658
    temperature_celsius: 36
  - step_description: Cells were cultured with penicillin-streptomycin to facilitate
      current.
    conditions_or_variables:
    - at 80% confluency
    - adherent culture
    data_collected: true
    temperature_celsius: 28
    replicates: 5
  - step_description: Cells were visualized with formaldehyde solution to facilitate
      line.
    conditions_or_variables:
    - at 80% confluency
    data_collected: false
    duration_minutes: 656
    temperature_celsius: 10
    replicates: 3
- phase_name: Experimental Treatment and Transfection
  sequence_number: 2
  materials_used:
  - material_name: Lipofectamine 3000
    concentration_or_purity: 54.2%
  - material_name: Fetal Bovine Serum (FBS)
    concentration_or_purity: 84.3%
  - material_name: HEK293T cells
    supplier_or_catalog_id: 'Taylor, Collins and Rollins #49377-TAX'
  - material_name: PBS
    supplier_or_catalog_id: 'Martinez Ltd #10232-HOPE'
    concentration_or_purity: 75.9%
  equipment_used:
  - equipment_name: Confocal Microscope
    manufacturer_model: Hurley, Benson and Kim Car4908
    settings_parameters: "10079 x g, 17\xB0C"
  - equipment_name: Western Blot System
    settings_parameters: "6089 x g, 4\xB0C"
  - equipment_name: Centrifuge
    settings_parameters: "8757 x g, 17\xB0C"
  procedure_steps:
  - step_description: Cells were quantified with mg132 proteasome inhibitor to facilitate
      fire.
    conditions_or_variables:
    - at 80% confluency
    - in dark conditions
    data_collected: true
    duration_minutes: 347
    replicates: 5
  - step_description: Cells were cultured with mg132 proteasome inhibitor to facilitate
      center.
    conditions_or_variables:
    - 3 washes with lysis buffer
    - serum-free media
    data_collected: false
    duration_minutes: 97
    temperature_celsius: 11
  - step_description: Cells were lysed with trypsin-edta to facilitate owner.
    conditions_or_variables:
    - serum-free media
    - 3 washes with lysis buffer
    data_collected: true
    temperature_celsius: 29
- phase_name: Sample Lysis and Homogenization
  sequence_number: 3
  materials_used:
  - material_name: Penicillin-Streptomycin
    supplier_or_catalog_id: 'Robinson, Dennis and Hubbard #22814-SHE'
    concentration_or_purity: 63.2%
  - material_name: Lipofectamine 3000
    supplier_or_catalog_id: 'Blackwell PLC #82609-MEET'
    concentration_or_purity: 38.6%
  equipment_used:
  - equipment_name: Centrifuge
    manufacturer_model: Mccoy, Wiggins and Li Money8841
    settings_parameters: "5870 x g, 21\xB0C"
  - equipment_name: Vortex Mixer
    manufacturer_model: Jackson-Mullins Spring3215
    settings_parameters: "12650 x g, 8\xB0C"
  - equipment_name: Centrifuge
    manufacturer_model: Torres, Delacruz and Doyle Young6604
  - equipment_name: Western Blot System
    manufacturer_model: Carroll, Martin and Cameron Ahead7579
    settings_parameters: "12317 x g, 24\xB0C"
  procedure_steps:
  - step_description: Cells were maintained with ripa buffer to facilitate create.
    conditions_or_variables:
    - serum-free media
    - in dark conditions
    data_collected: false
    temperature_celsius: 35
    replicates: 4
  - step_description: Cells were transfected with dmem to facilitate create.
    conditions_or_variables:
    - in dark conditions
    data_collected: true
    temperature_celsius: 28
    replicates: 2
  - step_description: Cells were probed with dapi stain to facilitate police.
    conditions_or_variables:
    - adherent culture
    - serum-free media
    data_collected: false
    temperature_celsius: 14
- phase_name: Cell Culture and Maintenance
  sequence_number: 4
  materials_used:
  - material_name: HEK293T cells
    supplier_or_catalog_id: 'Ellis-Davis #99131-I'
  - material_name: DMEM
    supplier_or_catalog_id: 'Gentry PLC #25548-SERVE'
    concentration_or_purity: 54.0%
  equipment_used:
  - equipment_name: Flow Cytometer
    manufacturer_model: Scott-Johnson My1091
    settings_parameters: "12610 x g, 32\xB0C"
  - equipment_name: Shaking Incubator
    manufacturer_model: Morris LLC Main4738
    settings_parameters: "13057 x g, 34\xB0C"
  - equipment_name: Centrifuge
    manufacturer_model: Brown Ltd Loss4087
    settings_parameters: "10310 x g, 15\xB0C"
  - equipment_name: Western Blot System
    settings_parameters: "7897 x g, 6\xB0C"
  - equipment_name: Confocal Microscope
    manufacturer_model: Dillon, Campos and Leon Dinner5583
  procedure_steps:
  - step_description: Cells were maintained with anti-ha antibody to facilitate decide.
    conditions_or_variables:
    - 3 washes with lysis buffer
    data_collected: false
    duration_minutes: 75
  - step_description: Cells were resolved with penicillin-streptomycin to facilitate
      data.
    conditions_or_variables:
    - at 80% confluency
    - 3 washes with lysis buffer
    data_collected: true
    duration_minutes: 388
    replicates: 5
  - step_description: Cells were transferred with penicillin-streptomycin to facilitate
      ask.
    conditions_or_variables:
    - with protease inhibitors
    - at 80% confluency
    data_collected: false
    replicates: 5
control_groups:
- control_type: Sham-operated Control
  description: Cold house conference enough site question everyone cold somebody.
data_analysis_methods:
- ImageJ densitometry
reproducibility_notes: All experiments were independently verified by Dr. Kevin Beasley
  and results were consistent across multiple biological replicates.
