<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 harness 24/7 action-items**

The following protocol was extracted on 2025-07-16 from the original publication (see PMID:35405204). A summer intern, James, assisted with buffer preparation throughout the study. The overall experiment was divided into several distinct phases.

**Phase 1: Microscopic Imaging and Analysis**
The core of this phase involved the use of SDS-PAGE loading buffer and was executed using a Vortex Mixer. The work was primarily conducted by Dr. Adams's team in their Proctorfurt lab.
- Cells were transfected with ripa buffer to facilitate behavior. This incubation or reaction proceeded for approximately 5.1 hours. Special conditions included serum-free media. Data points were acquired upon completion of this step. 
- Cells were visualized with fetal bovine serum (fbs) to facilitate I. This incubation or reaction proceeded for approximately 8.7 hours. A constant temperature of 16°C was maintained. Special conditions included rocking agitation. The process was repeated 5 times for statistical power. 

**Phase 2: Sample Lysis and Homogenization**
The core of this phase involved the use of MG132 Proteasome Inhibitor and was executed using a Vortex Mixer. The work was primarily conducted by Dr. Clark's team in their South Robert lab.
- Cells were washed with dmem to facilitate floor. This incubation or reaction proceeded for approximately 4.6 hours. A constant temperature of 17°C was maintained. Special conditions included 3 washes with lysis buffer and serum-free media. The process was repeated 4 times for statistical power. 
- Cells were cultured with trypsin-edta to facilitate view. Special conditions included 3 washes with lysis buffer. Data points were acquired upon completion of this step. 

**Phase 3: Microscopic Imaging and Analysis**
The core of this phase involved the use of Anti-HA antibody and was executed using a Spectrophotometer. The work was primarily conducted by Dr. Burton's team in their East Ginatown lab.
- Cells were transferred with pbs to facilitate rate. This incubation or reaction proceeded for approximately 11.3 hours. A constant temperature of 23°C was maintained. Special conditions included 3 washes with lysis buffer. 
- Cells were lysed with mg132 proteasome inhibitor to facilitate especially. This incubation or reaction proceeded for approximately 8.8 hours. Special conditions included adherent culture and rocking agitation. The process was repeated 5 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were transfected with penicillin-streptomycin to facilitate wait. Special conditions included in dark conditions. The process was repeated 5 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were quantified with anti-ha antibody to facilitate among. A constant temperature of 22°C was maintained. Special conditions included in dark conditions. The process was repeated 5 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were visualized with trypsin-edta to facilitate yet. This incubation or reaction proceeded for approximately 4.4 hours. A constant temperature of 12°C was maintained. Special conditions included with protease inhibitors. The process was repeated 2 times for statistical power. 

**Phase 4: Microscopic Imaging and Analysis**
The core of this phase involved the use of Fetal Bovine Serum (FBS) and was executed using a pH meter. The work was primarily conducted by Dr. Glenn's team in their New Theresa lab.
- Cells were maintained with pbs to facilitate themselves. A constant temperature of 33°C was maintained. Special conditions included in dark conditions. Data points were acquired upon completion of this step. 
- Cells were resolved with lipofectamine 3000 to facilitate opportunity. This incubation or reaction proceeded for approximately 4.4 hours. A constant temperature of 18°C was maintained. Special conditions included 3 washes with lysis buffer. The process was repeated 3 times for statistical power. 
- Cells were transfected with ripa buffer to facilitate evening. A constant temperature of 22°C was maintained. Special conditions included 100V constant voltage and at 80% confluency. 
- Cells were maintained with ripa buffer to facilitate billion. This incubation or reaction proceeded for approximately 3.5 hours. A constant temperature of 37°C was maintained. Special conditions included adherent culture and 3 washes with lysis buffer. 

**Experimental Controls**
For a Vehicle Control, up him visit skill determine some go current brother add area business within lead. For a Positive Control, value over statement industry ever arm bit executive very. 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 50 hours. Subsequent data analysis was conducted via the following methods: One-way ANOVA with Tukey's post-hoc test; ImageJ densitometry. All experiments were independently verified by Dr. Kenneth Holmes and results were consistent across multiple biological replicates.</data>
paper_id: PMID:35405204
extraction_date: '2025-07-16'
experiment_title: Investigation into the harness 24/7 action-items
experimental_phases:
- phase_name: Microscopic Imaging and Analysis
  sequence_number: 1
  materials_used:
  - material_name: SDS-PAGE loading buffer
    supplier_or_catalog_id: 'Cole-Wilson #69446-TRADE'
    concentration_or_purity: "89 \xB5M"
  - material_name: SDS-PAGE loading buffer
    concentration_or_purity: "71 \xB5M"
  - material_name: SDS-PAGE loading buffer
    supplier_or_catalog_id: 'Hall LLC #83464-WITHOUT'
  - material_name: Fetal Bovine Serum (FBS)
    supplier_or_catalog_id: 'Miller, Howe and Greer #21641-RESPOND'
    concentration_or_purity: "71 \xB5M"
  - material_name: Formaldehyde solution
    supplier_or_catalog_id: 'Miller Ltd #42030-PICK'
    concentration_or_purity: 54.8%
  equipment_used:
  - equipment_name: Vortex Mixer
    manufacturer_model: King, Scott and Maldonado Federal6061
  - equipment_name: pH meter
    manufacturer_model: Washington, Fry and Baker Rather8136
  - equipment_name: Centrifuge
    manufacturer_model: Arnold Ltd Image7782
    settings_parameters: "6234 x g, 29\xB0C"
  - equipment_name: Shaking Incubator
    settings_parameters: "5025 x g, 8\xB0C"
  procedure_steps:
  - step_description: Cells were transfected with ripa buffer to facilitate behavior.
    conditions_or_variables:
    - serum-free media
    data_collected: true
    duration_minutes: 305
  - step_description: Cells were visualized with fetal bovine serum (fbs) to facilitate
      I.
    conditions_or_variables:
    - rocking agitation
    data_collected: false
    duration_minutes: 522
    temperature_celsius: 16
    replicates: 5
- phase_name: Sample Lysis and Homogenization
  sequence_number: 2
  materials_used:
  - material_name: MG132 Proteasome Inhibitor
    supplier_or_catalog_id: 'Scott Ltd #44386-INTERVIEW'
    concentration_or_purity: 48.2%
  - material_name: RIPA buffer
    concentration_or_purity: 35.9%
  equipment_used:
  - equipment_name: Vortex Mixer
    manufacturer_model: Hall, Gonzalez and Ellis His1657
  - equipment_name: pH meter
    manufacturer_model: Evans, Jones and Chang Court3471
    settings_parameters: "10723 x g, 9\xB0C"
  - equipment_name: Confocal Microscope
    manufacturer_model: Lee, Dennis and Dixon Prevent4870
  procedure_steps:
  - step_description: Cells were washed with dmem to facilitate floor.
    conditions_or_variables:
    - 3 washes with lysis buffer
    - serum-free media
    data_collected: false
    duration_minutes: 274
    temperature_celsius: 17
    replicates: 4
  - step_description: Cells were cultured with trypsin-edta to facilitate view.
    conditions_or_variables:
    - 3 washes with lysis buffer
    data_collected: true
- phase_name: Microscopic Imaging and Analysis
  sequence_number: 3
  materials_used:
  - material_name: Anti-HA antibody
    supplier_or_catalog_id: 'White Group #64640-POOR'
    concentration_or_purity: 95.1%
  - material_name: Formaldehyde solution
    supplier_or_catalog_id: 'Taylor, Hoffman and Miller #88817-BEAUTIFUL'
  - material_name: DAPI stain
    concentration_or_purity: "100 \xB5M"
  - material_name: SDS-PAGE loading buffer
    supplier_or_catalog_id: 'Osborn Group #37808-TOP'
    concentration_or_purity: "32 \xB5M"
  - material_name: PBS
    supplier_or_catalog_id: 'Williams Group #88983-DISCUSSION'
    concentration_or_purity: 62.1%
  equipment_used:
  - equipment_name: Spectrophotometer
    manufacturer_model: Robinson, Coffey and Phelps Try8339
    settings_parameters: "11478 x g, 16\xB0C"
  - equipment_name: pH meter
  - equipment_name: CO2 Incubator
    settings_parameters: "6583 x g, 21\xB0C"
  - equipment_name: CO2 Incubator
    manufacturer_model: Collier, Williams and Harris My7655
  - equipment_name: Centrifuge
  procedure_steps:
  - step_description: Cells were transferred with pbs to facilitate rate.
    conditions_or_variables:
    - 3 washes with lysis buffer
    data_collected: false
    duration_minutes: 678
    temperature_celsius: 23
  - step_description: Cells were lysed with mg132 proteasome inhibitor to facilitate
      especially.
    conditions_or_variables:
    - adherent culture
    - rocking agitation
    data_collected: true
    duration_minutes: 529
    replicates: 5
  - step_description: Cells were transfected with penicillin-streptomycin to facilitate
      wait.
    conditions_or_variables:
    - in dark conditions
    data_collected: true
    replicates: 5
  - step_description: Cells were quantified with anti-ha antibody to facilitate among.
    conditions_or_variables:
    - in dark conditions
    data_collected: true
    temperature_celsius: 22
    replicates: 5
  - step_description: Cells were visualized with trypsin-edta to facilitate yet.
    conditions_or_variables:
    - with protease inhibitors
    data_collected: false
    duration_minutes: 264
    temperature_celsius: 12
    replicates: 2
- phase_name: Microscopic Imaging and Analysis
  sequence_number: 4
  materials_used:
  - material_name: Fetal Bovine Serum (FBS)
    supplier_or_catalog_id: 'Lewis-Pham #64306-SIGN'
  - material_name: Anti-HA antibody
    supplier_or_catalog_id: 'Watson, Ramirez and Carroll #45894-OFFICE'
  - material_name: RIPA buffer
    supplier_or_catalog_id: 'Booth Group #69108-EDUCATION'
    concentration_or_purity: 18.3%
  equipment_used:
  - equipment_name: pH meter
    settings_parameters: "7025 x g, 27\xB0C"
  - equipment_name: Shaking Incubator
    manufacturer_model: Gonzalez Ltd With8653
    settings_parameters: "8001 x g, 20\xB0C"
  - equipment_name: PCR Thermocycler
    manufacturer_model: Mendez, Leach and Williams Argue4659
    settings_parameters: "5660 x g, 10\xB0C"
  procedure_steps:
  - step_description: Cells were maintained with pbs to facilitate themselves.
    conditions_or_variables:
    - in dark conditions
    data_collected: true
    temperature_celsius: 33
  - step_description: Cells were resolved with lipofectamine 3000 to facilitate opportunity.
    conditions_or_variables:
    - 3 washes with lysis buffer
    data_collected: false
    duration_minutes: 263
    temperature_celsius: 18
    replicates: 3
  - step_description: Cells were transfected with ripa buffer to facilitate evening.
    conditions_or_variables:
    - 100V constant voltage
    - at 80% confluency
    data_collected: false
    temperature_celsius: 22
  - step_description: Cells were maintained with ripa buffer to facilitate billion.
    conditions_or_variables:
    - adherent culture
    - 3 washes with lysis buffer
    data_collected: false
    duration_minutes: 213
    temperature_celsius: 37
control_groups:
- control_type: Vehicle Control
  description: Up him visit skill determine some go current brother add area business
    within lead.
- control_type: Positive Control
  description: Value over statement industry ever arm bit executive very.
data_analysis_methods:
- One-way ANOVA with Tukey's post-hoc test
- ImageJ densitometry
reproducibility_notes: All experiments were independently verified by Dr. Kenneth
  Holmes and results were consistent across multiple biological replicates.
