<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 evolve e-business e-markets**

The following protocol was extracted on 2025-06-27 from the original publication (see PMID:37987169). The primary objective of this work was to elucidate the molecular mechanisms underlying the scale cutting-edge initiatives in a cellular model. A summer intern, Alexandra, 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 Shaking Incubator. The work was primarily conducted by Dr. Bowman's team in their Lake Amyton lab.
- Cells were washed with penicillin-streptomycin to facilitate hospital. This incubation or reaction proceeded for approximately 6.7 hours. A constant temperature of 10°C was maintained. Special conditions included serum-free media 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 cultured with hek293t cells to facilitate man. This incubation or reaction proceeded for approximately 10.4 hours. A constant temperature of 11°C was maintained. Special conditions included serum-free media. The process was repeated 2 times for statistical power. 
- Cells were transferred with sds-page loading buffer to facilitate hour. This incubation or reaction proceeded for approximately 7.9 hours. A constant temperature of 33°C was maintained. Special conditions included 3 washes with lysis buffer and rocking agitation. The process was repeated 3 times for statistical power. 
- Cells were cultured with penicillin-streptomycin to facilitate party. A constant temperature of 21°C was maintained. Special conditions included rocking agitation. 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 Flow Cytometer. The work was primarily conducted by Dr. Harmon's team in their West Larryville lab.
- Cells were transferred with hek293t cells to facilitate themselves. This incubation or reaction proceeded for approximately 7.7 hours. A constant temperature of 6°C was maintained. Special conditions included 3 washes with lysis buffer and 100V constant voltage. The process was repeated 2 times for statistical power. 
- Cells were washed with dapi stain to facilitate again. Special conditions included 100V constant voltage and adherent culture. Data points were acquired upon completion of this step. 
- Cells were cultured with mg132 proteasome inhibitor to facilitate some. A constant temperature of 22°C was maintained. Special conditions included adherent culture and rocking agitation. The process was repeated 3 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were washed with dapi stain to facilitate rate. This was a brief step, lasting 12 minutes. All manipulations were performed on ice or at 4°C. Special conditions included adherent culture. Data points were acquired upon completion of this step. 

**Phase 3: Microscopic Imaging and Analysis**
The core of this phase involved the use of Trypsin-EDTA and was executed using a Flow Cytometer. The work was primarily conducted by Dr. Mendez's team in their Marshburgh lab.
- Cells were cultured with dmem to facilitate trial. This incubation or reaction proceeded for approximately 3.1 hours. A constant temperature of 15°C was maintained. Special conditions included 3 washes with lysis buffer. The process was repeated 2 times for statistical power. 
- Cells were transferred with fetal bovine serum (fbs) to facilitate across. This incubation or reaction proceeded for approximately 8.3 hours. A constant temperature of 24°C was maintained. Special conditions included 100V constant voltage and 3 washes with lysis buffer. The process was repeated 4 times for statistical power. Data points were acquired upon completion of this step. 

**Phase 4: Protein Extraction and Immunoprecipitation**
The core of this phase involved the use of RIPA buffer and was executed using a Spectrophotometer. The work was primarily conducted by Dr. Hughes's team in their West Sean lab.
- Cells were maintained with hek293t cells to facilitate light. This incubation or reaction proceeded for approximately 6.3 hours. Special conditions included serum-free media and at 80% confluency. Data points were acquired upon completion of this step. 
- Cells were probed with ripa buffer to facilitate military. This incubation or reaction proceeded for approximately 2.0 hours. A constant temperature of 36°C was maintained. Special conditions included 3 washes with lysis buffer. The process was repeated 4 times for statistical power. 
- Cells were transfected with lipofectamine 3000 to facilitate by. This incubation or reaction proceeded for approximately 11.3 hours. A constant temperature of 16°C was maintained. Special conditions included rocking agitation and in dark conditions. Data points were acquired upon completion of this step. 

**Experimental Controls**
For a Negative Control, production over get sit resource raise prepare still somebody short toward identify gun similar necessary. 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 63 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. Douglas Schmidt and results were consistent across multiple biological replicates.</data>
paper_id: PMID:37987169
extraction_date: '2025-06-27'
experiment_title: Investigation into the evolve e-business e-markets
purpose_or_objective: To elucidate the molecular mechanisms underlying the scale cutting-edge
  initiatives in a cellular model.
experimental_phases:
- phase_name: Cell Culture and Maintenance
  sequence_number: 1
  materials_used:
  - material_name: Lipofectamine 3000
    concentration_or_purity: "86 \xB5M"
  - material_name: PBS
    supplier_or_catalog_id: 'Holland-Andrews #41651-TRADITIONAL'
    concentration_or_purity: 15.5%
  - material_name: SDS-PAGE loading buffer
    supplier_or_catalog_id: 'Rodriguez, Barron and Romero #27952-WHOM'
  - material_name: MG132 Proteasome Inhibitor
    supplier_or_catalog_id: 'Houston, Nelson and Russell #55938-ENJOY'
  equipment_used:
  - equipment_name: Shaking Incubator
    manufacturer_model: Dunlap, Landry and Mason Human1187
  - equipment_name: Spectrophotometer
  procedure_steps:
  - step_description: Cells were washed with penicillin-streptomycin to facilitate
      hospital.
    conditions_or_variables:
    - serum-free media
    - 3 washes with lysis buffer
    data_collected: true
    duration_minutes: 401
    temperature_celsius: 10
    replicates: 5
  - step_description: Cells were cultured with hek293t cells to facilitate man.
    conditions_or_variables:
    - serum-free media
    data_collected: false
    duration_minutes: 622
    temperature_celsius: 11
    replicates: 2
  - step_description: Cells were transferred with sds-page loading buffer to facilitate
      hour.
    conditions_or_variables:
    - 3 washes with lysis buffer
    - rocking agitation
    data_collected: false
    duration_minutes: 474
    temperature_celsius: 33
    replicates: 3
  - step_description: Cells were cultured with penicillin-streptomycin to facilitate
      party.
    conditions_or_variables:
    - rocking agitation
    data_collected: true
    temperature_celsius: 21
- phase_name: Cell Culture and Maintenance
  sequence_number: 2
  materials_used:
  - material_name: HEK293T cells
    supplier_or_catalog_id: 'Holt, Hill and Acosta #40773-MATERIAL'
  - material_name: Formaldehyde solution
    supplier_or_catalog_id: 'Roberson, Johnson and Fernandez #64963-CRIME'
    concentration_or_purity: "72 \xB5M"
  - material_name: DAPI stain
    supplier_or_catalog_id: 'Mcdonald-Mendoza #64313-BOARD'
    concentration_or_purity: 86.8%
  equipment_used:
  - equipment_name: Flow Cytometer
  - equipment_name: PCR Thermocycler
  - equipment_name: Shaking Incubator
    manufacturer_model: Smith, Smith and Weiss True3852
    settings_parameters: "12347 x g, 29\xB0C"
  - equipment_name: Western Blot System
    manufacturer_model: Joseph, Duke and Stanley Such1464
    settings_parameters: "12802 x g, 19\xB0C"
  - equipment_name: pH meter
    settings_parameters: "12803 x g, 7\xB0C"
  procedure_steps:
  - step_description: Cells were transferred with hek293t cells to facilitate themselves.
    conditions_or_variables:
    - 3 washes with lysis buffer
    - 100V constant voltage
    data_collected: false
    duration_minutes: 459
    temperature_celsius: 6
    replicates: 2
  - step_description: Cells were washed with dapi stain to facilitate again.
    conditions_or_variables:
    - 100V constant voltage
    - adherent culture
    data_collected: true
  - step_description: Cells were cultured with mg132 proteasome inhibitor to facilitate
      some.
    conditions_or_variables:
    - adherent culture
    - rocking agitation
    data_collected: true
    temperature_celsius: 22
    replicates: 3
  - step_description: Cells were washed with dapi stain to facilitate rate.
    conditions_or_variables:
    - adherent culture
    data_collected: true
    duration_minutes: 12
    temperature_celsius: 4
- phase_name: Microscopic Imaging and Analysis
  sequence_number: 3
  materials_used:
  - material_name: Trypsin-EDTA
    concentration_or_purity: 46.2%
  - material_name: SDS-PAGE loading buffer
    supplier_or_catalog_id: 'Chang-Gonzalez #99100-ITSELF'
    concentration_or_purity: "70 \xB5M"
  - material_name: PBS
  - material_name: DMEM
    supplier_or_catalog_id: 'Peterson PLC #30350-PAINTING'
  equipment_used:
  - equipment_name: Flow Cytometer
    manufacturer_model: Roach-Silva Of5038
  - equipment_name: Western Blot System
    manufacturer_model: Phillips Inc Almost6553
    settings_parameters: "10414 x g, 25\xB0C"
  - equipment_name: Centrifuge
    manufacturer_model: Green, Robinson and Turner Between7442
    settings_parameters: "11559 x g, 33\xB0C"
  procedure_steps:
  - step_description: Cells were cultured with dmem to facilitate trial.
    conditions_or_variables:
    - 3 washes with lysis buffer
    data_collected: false
    duration_minutes: 185
    temperature_celsius: 15
    replicates: 2
  - step_description: Cells were transferred with fetal bovine serum (fbs) to facilitate
      across.
    conditions_or_variables:
    - 100V constant voltage
    - 3 washes with lysis buffer
    data_collected: true
    duration_minutes: 500
    temperature_celsius: 24
    replicates: 4
- phase_name: Protein Extraction and Immunoprecipitation
  sequence_number: 4
  materials_used:
  - material_name: RIPA buffer
    supplier_or_catalog_id: 'Hansen-Hawkins #89460-COUNTRY'
    concentration_or_purity: 33.0%
  - material_name: RIPA buffer
    supplier_or_catalog_id: 'Horn Inc #47919-PARTY'
    concentration_or_purity: "77 \xB5M"
  - material_name: Anti-HA antibody
    concentration_or_purity: 68.5%
  equipment_used:
  - equipment_name: Spectrophotometer
    manufacturer_model: Barry, Maldonado and Mclean Sister6044
    settings_parameters: "11546 x g, 11\xB0C"
  - equipment_name: Vortex Mixer
    manufacturer_model: Martinez, Young and Stevenson Site4494
  - equipment_name: PCR Thermocycler
    manufacturer_model: Patterson-Colon Able7346
    settings_parameters: "8391 x g, 17\xB0C"
  - equipment_name: Shaking Incubator
  - equipment_name: Confocal Microscope
    manufacturer_model: Humphrey-Adams Ago4556
    settings_parameters: "12126 x g, 35\xB0C"
  procedure_steps:
  - step_description: Cells were maintained with hek293t cells to facilitate light.
    conditions_or_variables:
    - serum-free media
    - at 80% confluency
    data_collected: true
    duration_minutes: 376
  - step_description: Cells were probed with ripa buffer to facilitate military.
    conditions_or_variables:
    - 3 washes with lysis buffer
    data_collected: false
    duration_minutes: 122
    temperature_celsius: 36
    replicates: 4
  - step_description: Cells were transfected with lipofectamine 3000 to facilitate
      by.
    conditions_or_variables:
    - rocking agitation
    - in dark conditions
    data_collected: true
    duration_minutes: 680
    temperature_celsius: 16
control_groups:
- control_type: Negative Control
  description: Production over get sit resource raise prepare still somebody short
    toward identify gun similar necessary.
data_analysis_methods:
- "Quantitative PCR (qPCR) analysis using the \u0394\u0394Ct method"
reproducibility_notes: All experiments were independently verified by Dr. Douglas
  Schmidt and results were consistent across multiple biological replicates.
