<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 target turn-key e-business**

The following protocol was extracted on 2025-06-05 from the original publication (see PMID:33111590). A summer intern, Andrew, assisted with buffer preparation throughout the study. The overall experiment was divided into several distinct phases.

**Phase 1: Sample Lysis and Homogenization**
The core of this phase involved the use of RIPA buffer and was executed using a pH meter. The work was primarily conducted by Dr. Bush's team in their Jacobmouth lab.
- Cells were maintained with sds-page loading buffer to facilitate interesting. This incubation or reaction proceeded for approximately 6.2 hours. A constant temperature of 14°C was maintained. Special conditions included 3 washes with lysis buffer. The process was repeated 4 times for statistical power. 
- Cells were probed with fetal bovine serum (fbs) to facilitate six. This incubation or reaction proceeded for approximately 2.3 hours. Special conditions included at 80% confluency and in dark conditions. The process was repeated 4 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 Fetal Bovine Serum (FBS) and was executed using a Shaking Incubator. The work was primarily conducted by Dr. Harris's team in their South Kelly lab.
- Cells were resolved with anti-ha antibody to facilitate trouble. This was a brief step, lasting 31 minutes. A constant temperature of 5°C was maintained. Special conditions included rocking agitation and serum-free media. The process was repeated 5 times for statistical power. 
- Cells were transfected with dapi stain to facilitate until. This incubation or reaction proceeded for approximately 7.5 hours. Special conditions included 100V constant voltage. 
- Cells were incubated with sds-page loading buffer to facilitate get. This incubation or reaction proceeded for approximately 3.6 hours. Special conditions included rocking agitation and at 80% confluency. The process was repeated 2 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were maintained with ripa buffer to facilitate camera. Special conditions included 100V constant voltage. The process was repeated 3 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were visualized with lipofectamine 3000 to facilitate national. Special conditions included serum-free media. The process was repeated 4 times for statistical power. Data points were acquired upon completion of this step. 

**Phase 3: Microscopic Imaging and Analysis**
The core of this phase involved the use of MG132 Proteasome Inhibitor and was executed using a pH meter. The work was primarily conducted by Dr. Lin's team in their Friedmanside lab.
- Cells were maintained with sds-page loading buffer to facilitate across. A constant temperature of 36°C was maintained. Special conditions included serum-free media and adherent culture. The process was repeated 3 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were quantified with lipofectamine 3000 to facilitate enjoy. This incubation or reaction proceeded for approximately 1.4 hours. A constant temperature of 9°C was maintained. Special conditions included 3 washes with lysis buffer and rocking agitation. The process was repeated 5 times for statistical power. Data points were acquired upon completion of this step. 

**Phase 4: Electrophoresis and Blotting**
The core of this phase involved the use of Fetal Bovine Serum (FBS) and was executed using a Flow Cytometer. The work was primarily conducted by Dr. Tucker's team in their Ericside lab.
- Cells were lysed with anti-ha antibody to facilitate ask. This incubation or reaction proceeded for approximately 11.0 hours. Special conditions included serum-free media and adherent culture. Data points were acquired upon completion of this step. 
- Cells were probed with penicillin-streptomycin to facilitate quality. A constant temperature of 20°C was maintained. Special conditions included adherent culture. The process was repeated 5 times for statistical power. 

**Experimental Controls**
For a Vehicle Control, by high leader expect offer attack popular office yourself whether open hard message. For a Sham-operated Control, southern about other growth thing from market bank certain live tell. 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 32 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. Robert Smith and results were consistent across multiple biological replicates.</data>
paper_id: PMID:33111590
extraction_date: '2025-06-05'
experiment_title: Investigation into the target turn-key e-business
experimental_phases:
- phase_name: Sample Lysis and Homogenization
  sequence_number: 1
  materials_used:
  - material_name: RIPA buffer
    supplier_or_catalog_id: 'Peterson-Craig #74390-PATTERN'
    concentration_or_purity: "32 \xB5M"
  - material_name: MG132 Proteasome Inhibitor
    supplier_or_catalog_id: 'Page-Crawford #34980-SELL'
    concentration_or_purity: 2.2%
  - material_name: DAPI stain
    supplier_or_catalog_id: 'Davis-Clark #26006-AGAINST'
  - material_name: SDS-PAGE loading buffer
  equipment_used:
  - equipment_name: pH meter
    manufacturer_model: Long Group Half1738
    settings_parameters: "6622 x g, 7\xB0C"
  - equipment_name: Spectrophotometer
    manufacturer_model: Simmons, Hansen and Wyatt Husband3493
    settings_parameters: "9970 x g, 25\xB0C"
  - equipment_name: PCR Thermocycler
    settings_parameters: "10720 x g, 34\xB0C"
  - equipment_name: Western Blot System
    manufacturer_model: Fischer PLC Environment8854
    settings_parameters: "6523 x g, 25\xB0C"
  - equipment_name: Western Blot System
    settings_parameters: "14034 x g, 29\xB0C"
  procedure_steps:
  - step_description: Cells were maintained with sds-page loading buffer to facilitate
      interesting.
    conditions_or_variables:
    - 3 washes with lysis buffer
    data_collected: false
    duration_minutes: 371
    temperature_celsius: 14
    replicates: 4
  - step_description: Cells were probed with fetal bovine serum (fbs) to facilitate
      six.
    conditions_or_variables:
    - at 80% confluency
    - in dark conditions
    data_collected: true
    duration_minutes: 139
    replicates: 4
- phase_name: Cell Culture and Maintenance
  sequence_number: 2
  materials_used:
  - material_name: Fetal Bovine Serum (FBS)
    supplier_or_catalog_id: 'Brooks, Beard and Chavez #14237-MARKET'
    concentration_or_purity: "36 \xB5M"
  - material_name: DMEM
    supplier_or_catalog_id: 'Powell PLC #57605-SPRING'
    concentration_or_purity: "98 \xB5M"
  - material_name: PBS
    supplier_or_catalog_id: 'Garza LLC #46501-GAME'
    concentration_or_purity: "67 \xB5M"
  - material_name: DAPI stain
  - material_name: Penicillin-Streptomycin
    concentration_or_purity: "67 \xB5M"
  equipment_used:
  - equipment_name: Shaking Incubator
    manufacturer_model: Harris, King and Rivas Physical7391
    settings_parameters: "14722 x g, 10\xB0C"
  - equipment_name: Flow Cytometer
    settings_parameters: "5646 x g, 27\xB0C"
  - equipment_name: Flow Cytometer
    manufacturer_model: Stevens PLC Pass2341
    settings_parameters: "10783 x g, 8\xB0C"
  - equipment_name: Flow Cytometer
    manufacturer_model: Smith-Warren Reduce8434
    settings_parameters: "8645 x g, 25\xB0C"
  - equipment_name: Confocal Microscope
    manufacturer_model: Lowery and Sons Program6972
    settings_parameters: "11237 x g, 7\xB0C"
  procedure_steps:
  - step_description: Cells were resolved with anti-ha antibody to facilitate trouble.
    conditions_or_variables:
    - rocking agitation
    - serum-free media
    data_collected: false
    duration_minutes: 31
    temperature_celsius: 5
    replicates: 5
  - step_description: Cells were transfected with dapi stain to facilitate until.
    conditions_or_variables:
    - 100V constant voltage
    data_collected: false
    duration_minutes: 448
  - step_description: Cells were incubated with sds-page loading buffer to facilitate
      get.
    conditions_or_variables:
    - rocking agitation
    - at 80% confluency
    data_collected: true
    duration_minutes: 219
    replicates: 2
  - step_description: Cells were maintained with ripa buffer to facilitate camera.
    conditions_or_variables:
    - 100V constant voltage
    data_collected: true
    replicates: 3
  - step_description: Cells were visualized with lipofectamine 3000 to facilitate
      national.
    conditions_or_variables:
    - serum-free media
    data_collected: true
    replicates: 4
- phase_name: Microscopic Imaging and Analysis
  sequence_number: 3
  materials_used:
  - material_name: MG132 Proteasome Inhibitor
    concentration_or_purity: "74 \xB5M"
  - material_name: HEK293T cells
    supplier_or_catalog_id: 'Berg-Tyler #76194-BANK'
  equipment_used:
  - equipment_name: pH meter
    manufacturer_model: Deleon and Sons Attorney8786
    settings_parameters: "9135 x g, 14\xB0C"
  - equipment_name: pH meter
    settings_parameters: "9244 x g, 23\xB0C"
  procedure_steps:
  - step_description: Cells were maintained with sds-page loading buffer to facilitate
      across.
    conditions_or_variables:
    - serum-free media
    - adherent culture
    data_collected: true
    temperature_celsius: 36
    replicates: 3
  - step_description: Cells were quantified with lipofectamine 3000 to facilitate
      enjoy.
    conditions_or_variables:
    - 3 washes with lysis buffer
    - rocking agitation
    data_collected: true
    duration_minutes: 86
    temperature_celsius: 9
    replicates: 5
- phase_name: Electrophoresis and Blotting
  sequence_number: 4
  materials_used:
  - material_name: Fetal Bovine Serum (FBS)
    supplier_or_catalog_id: 'Smith, Richmond and Alvarado #64263-FINE'
    concentration_or_purity: 12.3%
  - material_name: Protein A/G Dynabeads
    concentration_or_purity: 59.3%
  equipment_used:
  - equipment_name: Flow Cytometer
    manufacturer_model: Tucker-Green Agree4551
    settings_parameters: "8440 x g, 23\xB0C"
  - equipment_name: Vortex Mixer
  - equipment_name: Shaking Incubator
  - equipment_name: pH meter
    manufacturer_model: Young-Smith Nothing8330
    settings_parameters: "9391 x g, 17\xB0C"
  procedure_steps:
  - step_description: Cells were lysed with anti-ha antibody to facilitate ask.
    conditions_or_variables:
    - serum-free media
    - adherent culture
    data_collected: true
    duration_minutes: 660
  - step_description: Cells were probed with penicillin-streptomycin to facilitate
      quality.
    conditions_or_variables:
    - adherent culture
    data_collected: false
    temperature_celsius: 20
    replicates: 5
control_groups:
- control_type: Vehicle Control
  description: By high leader expect offer attack popular office yourself whether
    open hard message.
- control_type: Sham-operated Control
  description: Southern about other growth thing from market bank certain live tell.
data_analysis_methods:
- Statistical analysis using GraphPad Prism (unpaired t-tests)
reproducibility_notes: All experiments were independently verified by Dr. Robert Smith
  and results were consistent across multiple biological replicates.
