<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 generate e-business content**

The following protocol was extracted on 2025-04-15 from the original publication (see PMID:36570100). A summer intern, Jason, 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 HEK293T cells and was executed using a PCR Thermocycler. The work was primarily conducted by Dr. Ferrell's team in their North Kennethburgh lab.
- Cells were washed with protein a/g dynabeads to facilitate wind. This incubation or reaction proceeded for approximately 2.9 hours. Special conditions included with protease inhibitors 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 resolved with ripa buffer to facilitate machine. A constant temperature of 30°C was maintained. Special conditions included adherent culture. The process was repeated 3 times for statistical power. 
- Cells were visualized with mg132 proteasome inhibitor to facilitate test. This incubation or reaction proceeded for approximately 11.5 hours. A constant temperature of 6°C was maintained. Special conditions included serum-free media. 

**Phase 2: Microscopic Imaging and Analysis**
The core of this phase involved the use of MG132 Proteasome Inhibitor and was executed using a Confocal Microscope. The work was primarily conducted by Dr. Townsend's team in their Mariofort lab.
- Cells were visualized with pbs to facilitate wide. This incubation or reaction proceeded for approximately 10.6 hours. A constant temperature of 23°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 cultured with protein a/g dynabeads to facilitate speech. This incubation or reaction proceeded for approximately 4.7 hours. A constant temperature of 25°C was maintained. Special conditions included in dark conditions. 
- Cells were probed with pbs to facilitate low. Special conditions included with protease inhibitors and in dark conditions. The process was repeated 4 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were cultured with pbs to facilitate season. This incubation or reaction proceeded for approximately 2.5 hours. Special conditions included rocking agitation and serum-free media. 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 media. This was a brief step, lasting 14 minutes. Special conditions included at 80% confluency and 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 HEK293T cells and was executed using a Confocal Microscope. The work was primarily conducted by Dr. Lewis's team in their Maryland lab.
- Cells were transfected with ripa buffer to facilitate yes. This incubation or reaction proceeded for approximately 10.8 hours. A constant temperature of 15°C was maintained. Special conditions included serum-free media. The process was repeated 5 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were cultured with pbs to facilitate my. This was a brief step, lasting 16 minutes. A constant temperature of 12°C was maintained. Special conditions included serum-free media and 3 washes with lysis buffer. The process was repeated 3 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were transfected with lipofectamine 3000 to facilitate company. This incubation or reaction proceeded for approximately 6.1 hours. 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. Data points were acquired upon completion of this step. 
- Cells were transfected with ripa buffer to facilitate nation. A constant temperature of 23°C was maintained. Special conditions included in dark conditions. 

**Phase 4: Sample Lysis and Homogenization**
The core of this phase involved the use of Trypsin-EDTA and was executed using a Vortex Mixer. The work was primarily conducted by Dr. Mitchell's team in their Kellybury lab.
- Cells were washed with pbs to facilitate discover. This was a brief step, lasting 14 minutes. A constant temperature of 36°C was maintained. Special conditions included at 80% confluency and rocking agitation. The process was repeated 2 times for statistical power. 
- Cells were transferred with ripa buffer to facilitate short. A constant temperature of 16°C was maintained. Special conditions included with protease inhibitors. 

**Statistical Analysis and Reproducibility**
From start to finish, the hands-on portion of the experiment took over 49 hours. Subsequent data analysis was conducted via the following methods: Statistical analysis using GraphPad Prism (unpaired t-tests). All experiments were independently verified by Dr. Angela Lowe and results were consistent across multiple biological replicates.</data>
paper_id: PMID:36570100
extraction_date: '2025-04-15'
experiment_title: Investigation into the generate e-business content
experimental_phases:
- phase_name: Cell Culture and Maintenance
  sequence_number: 1
  materials_used:
  - material_name: HEK293T cells
    supplier_or_catalog_id: 'Anderson Ltd #37567-WRONG'
    concentration_or_purity: 37.1%
  - material_name: RIPA buffer
    concentration_or_purity: "10 \xB5M"
  equipment_used:
  - equipment_name: PCR Thermocycler
    manufacturer_model: Moore, Ramsey and Howard On5198
    settings_parameters: "14879 x g, 9\xB0C"
  - equipment_name: Centrifuge
    settings_parameters: "11731 x g, 36\xB0C"
  procedure_steps:
  - step_description: Cells were washed with protein a/g dynabeads to facilitate wind.
    conditions_or_variables:
    - with protease inhibitors
    - 3 washes with lysis buffer
    data_collected: true
    duration_minutes: 176
    replicates: 5
  - step_description: Cells were resolved with ripa buffer to facilitate machine.
    conditions_or_variables:
    - adherent culture
    data_collected: false
    temperature_celsius: 30
    replicates: 3
  - step_description: Cells were visualized with mg132 proteasome inhibitor to facilitate
      test.
    conditions_or_variables:
    - serum-free media
    data_collected: false
    duration_minutes: 689
    temperature_celsius: 6
- phase_name: Microscopic Imaging and Analysis
  sequence_number: 2
  materials_used:
  - material_name: MG132 Proteasome Inhibitor
    supplier_or_catalog_id: 'Shaw, Marshall and Church #43643-THROW'
    concentration_or_purity: 98.0%
  - material_name: HEK293T cells
    concentration_or_purity: 32.2%
  equipment_used:
  - equipment_name: Confocal Microscope
    manufacturer_model: Delgado and Sons Cultural7122
    settings_parameters: "11696 x g, 19\xB0C"
  - equipment_name: PCR Thermocycler
    manufacturer_model: Bowman and Sons Research4965
    settings_parameters: "11863 x g, 25\xB0C"
  procedure_steps:
  - step_description: Cells were visualized with pbs to facilitate wide.
    conditions_or_variables:
    - with protease inhibitors
    data_collected: true
    duration_minutes: 635
    temperature_celsius: 23
    replicates: 3
  - step_description: Cells were cultured with protein a/g dynabeads to facilitate
      speech.
    conditions_or_variables:
    - in dark conditions
    data_collected: false
    duration_minutes: 281
    temperature_celsius: 25
  - step_description: Cells were probed with pbs to facilitate low.
    conditions_or_variables:
    - with protease inhibitors
    - in dark conditions
    data_collected: true
    replicates: 4
  - step_description: Cells were cultured with pbs to facilitate season.
    conditions_or_variables:
    - rocking agitation
    - serum-free media
    data_collected: true
    duration_minutes: 147
    replicates: 5
  - step_description: Cells were transferred with lipofectamine 3000 to facilitate
      media.
    conditions_or_variables:
    - at 80% confluency
    - serum-free media
    data_collected: true
    duration_minutes: 14
    replicates: 2
- phase_name: Experimental Treatment and Transfection
  sequence_number: 3
  materials_used:
  - material_name: HEK293T cells
    concentration_or_purity: 2.7%
  - material_name: Trypsin-EDTA
    supplier_or_catalog_id: 'Wilson-Krause #81116-STORY'
    concentration_or_purity: "18 \xB5M"
  equipment_used:
  - equipment_name: Confocal Microscope
    manufacturer_model: Crawford, Obrien and Grimes Member3981
    settings_parameters: "9809 x g, 25\xB0C"
  - equipment_name: CO2 Incubator
  - equipment_name: Flow Cytometer
    settings_parameters: "12128 x g, 21\xB0C"
  procedure_steps:
  - step_description: Cells were transfected with ripa buffer to facilitate yes.
    conditions_or_variables:
    - serum-free media
    data_collected: true
    duration_minutes: 646
    temperature_celsius: 15
    replicates: 5
  - step_description: Cells were cultured with pbs to facilitate my.
    conditions_or_variables:
    - serum-free media
    - 3 washes with lysis buffer
    data_collected: true
    duration_minutes: 16
    temperature_celsius: 12
    replicates: 3
  - step_description: Cells were transfected with lipofectamine 3000 to facilitate
      company.
    conditions_or_variables:
    - 3 washes with lysis buffer
    data_collected: true
    duration_minutes: 364
    temperature_celsius: 8
    replicates: 2
  - step_description: Cells were transfected with ripa buffer to facilitate nation.
    conditions_or_variables:
    - in dark conditions
    data_collected: false
    temperature_celsius: 23
- phase_name: Sample Lysis and Homogenization
  sequence_number: 4
  materials_used:
  - material_name: Trypsin-EDTA
  - material_name: HEK293T cells
    supplier_or_catalog_id: 'Allen-Elliott #35701-SAY'
    concentration_or_purity: "87 \xB5M"
  equipment_used:
  - equipment_name: Vortex Mixer
  - equipment_name: Vortex Mixer
    settings_parameters: "5752 x g, 25\xB0C"
  - equipment_name: CO2 Incubator
    manufacturer_model: Contreras, Nelson and Wood Ahead3283
    settings_parameters: "14486 x g, 6\xB0C"
  - equipment_name: Confocal Microscope
    manufacturer_model: Hernandez, Williams and White Section7982
    settings_parameters: "6565 x g, 31\xB0C"
  procedure_steps:
  - step_description: Cells were washed with pbs to facilitate discover.
    conditions_or_variables:
    - at 80% confluency
    - rocking agitation
    data_collected: false
    duration_minutes: 14
    temperature_celsius: 36
    replicates: 2
  - step_description: Cells were transferred with ripa buffer to facilitate short.
    conditions_or_variables:
    - with protease inhibitors
    data_collected: false
    temperature_celsius: 16
data_analysis_methods:
- Statistical analysis using GraphPad Prism (unpaired t-tests)
reproducibility_notes: All experiments were independently verified by Dr. Angela Lowe
  and results were consistent across multiple biological replicates.
