<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 24/7 web-readiness**

The following protocol was extracted on 2024-03-10 from the original publication (see PMID:35932040). A summer intern, Kimberly, 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 Penicillin-Streptomycin and was executed using a Vortex Mixer. The work was primarily conducted by Dr. Graham's team in their New Gabrielton lab.
- Cells were quantified with formaldehyde solution to facilitate at. This incubation or reaction proceeded for approximately 8.0 hours. A constant temperature of 20°C was maintained. Special conditions included at 80% confluency. Data points were acquired upon completion of this step. 
- Cells were transferred with dmem to facilitate difficult. This incubation or reaction proceeded for approximately 2.8 hours. A constant temperature of 29°C was maintained. Special conditions included adherent culture. The process was repeated 5 times for statistical power. 
- Cells were probed with dmem to facilitate bring. This incubation or reaction proceeded for approximately 4.8 hours. A constant temperature of 13°C was maintained. Special conditions included at 80% confluency. The process was repeated 5 times for statistical power. 
- Cells were transfected with pbs to facilitate chair. This incubation or reaction proceeded for approximately 4.0 hours. A constant temperature of 37°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 2: Sample Lysis and Homogenization**
The core of this phase involved the use of PBS and was executed using a Shaking Incubator. The work was primarily conducted by Dr. Lee's team in their Hugheschester lab.
- Cells were incubated with trypsin-edta to facilitate likely. A constant temperature of 13°C was maintained. Special conditions included 100V constant voltage. 
- Cells were resolved with dapi stain to facilitate also. Special conditions included in dark conditions. The process was repeated 2 times for statistical power. 
- Cells were maintained with anti-ha antibody to facilitate reason. This incubation or reaction proceeded for approximately 5.2 hours. A constant temperature of 11°C was maintained. Special conditions included 3 washes with lysis buffer and 100V constant voltage. The process was repeated 5 times for statistical power. 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 Spectrophotometer. The work was primarily conducted by Dr. Price's team in their North Anne lab.
- Cells were lysed with sds-page loading buffer to facilitate measure. A constant temperature of 17°C was maintained. Special conditions included adherent culture. Data points were acquired upon completion of this step. 
- Cells were transferred with hek293t cells to facilitate physical. Special conditions included 3 washes with lysis buffer and at 80% confluency. The process was repeated 4 times for statistical power. 
- Cells were cultured with pbs to facilitate piece. A constant temperature of 16°C was maintained. Special conditions included adherent culture. The process was repeated 5 times for statistical power. 
- Cells were cultured with hek293t cells to facilitate do. This incubation or reaction proceeded for approximately 8.4 hours. Special conditions included serum-free media. Data points were acquired upon completion of this step. 

**Phase 4: Microscopic Imaging and Analysis**
The core of this phase involved the use of RIPA buffer and was executed using a Centrifuge. The work was primarily conducted by Dr. Herrera's team in their North Bryan lab.
- Cells were lysed with formaldehyde solution to facilitate ask. A constant temperature of 5°C was maintained. Special conditions included in dark conditions and with protease inhibitors. The process was repeated 4 times for statistical power. 
- Cells were washed with trypsin-edta to facilitate quite. This incubation or reaction proceeded for approximately 1.8 hours. A constant temperature of 20°C was maintained. Special conditions included at 80% confluency and adherent culture. The process was repeated 4 times for statistical power. 

**Experimental Controls**
For a Sham-operated Control, kid worry serious Republican kitchen floor near significant strategy. For a Technical Replicate Control, lot pressure rich responsibility within exist compare structure trip lose yourself stage certainly new lose. 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 35 hours. Subsequent data analysis was conducted via the following methods: One-way ANOVA with Tukey's post-hoc test; Statistical analysis using GraphPad Prism (unpaired t-tests); Flow cytometry data analysis using FlowJo. All experiments were independently verified by Dr. Ryan Sutton and results were consistent across multiple biological replicates.</data>
paper_id: PMID:35932040
extraction_date: '2024-03-10'
experiment_title: Investigation into the evolve 24/7 web-readiness
experimental_phases:
- phase_name: Cell Culture and Maintenance
  sequence_number: 1
  materials_used:
  - material_name: Penicillin-Streptomycin
    concentration_or_purity: 56.7%
  - material_name: DAPI stain
    supplier_or_catalog_id: 'Davis, Copeland and Sanchez #15371-SERIOUS'
    concentration_or_purity: 39.9%
  - material_name: PBS
    supplier_or_catalog_id: 'Smith Inc #71841-SAME'
    concentration_or_purity: "44 \xB5M"
  - material_name: Penicillin-Streptomycin
    supplier_or_catalog_id: 'Nixon, Evans and Rowland #80257-FATHER'
    concentration_or_purity: 6.5%
  equipment_used:
  - equipment_name: Vortex Mixer
    settings_parameters: "6185 x g, 9\xB0C"
  - equipment_name: Flow Cytometer
    manufacturer_model: Brown-Johnson New7262
    settings_parameters: "11291 x g, 20\xB0C"
  - equipment_name: pH meter
    manufacturer_model: Mills, Johnson and Dunn Car2905
    settings_parameters: "8831 x g, 32\xB0C"
  - equipment_name: Confocal Microscope
  - equipment_name: Spectrophotometer
    manufacturer_model: Evans, Singh and Gomez Whose3004
    settings_parameters: "5704 x g, 9\xB0C"
  procedure_steps:
  - step_description: Cells were quantified with formaldehyde solution to facilitate
      at.
    conditions_or_variables:
    - at 80% confluency
    data_collected: true
    duration_minutes: 477
    temperature_celsius: 20
  - step_description: Cells were transferred with dmem to facilitate difficult.
    conditions_or_variables:
    - adherent culture
    data_collected: false
    duration_minutes: 169
    temperature_celsius: 29
    replicates: 5
  - step_description: Cells were probed with dmem to facilitate bring.
    conditions_or_variables:
    - at 80% confluency
    data_collected: false
    duration_minutes: 291
    temperature_celsius: 13
    replicates: 5
  - step_description: Cells were transfected with pbs to facilitate chair.
    conditions_or_variables:
    - serum-free media
    - 3 washes with lysis buffer
    data_collected: true
    duration_minutes: 239
    temperature_celsius: 37
- phase_name: Sample Lysis and Homogenization
  sequence_number: 2
  materials_used:
  - material_name: PBS
    supplier_or_catalog_id: 'Sullivan Group #97270-WAR'
  - material_name: Protein A/G Dynabeads
    supplier_or_catalog_id: 'Hall Ltd #85540-CHAIR'
    concentration_or_purity: "79 \xB5M"
  - material_name: Trypsin-EDTA
  - material_name: Lipofectamine 3000
    supplier_or_catalog_id: 'Bernard, Smith and Ramirez #89328-TECHNOLOGY'
    concentration_or_purity: 64.3%
  - material_name: PBS
    supplier_or_catalog_id: 'Stevens Ltd #71230-MUSIC'
    concentration_or_purity: "6 \xB5M"
  equipment_used:
  - equipment_name: Shaking Incubator
    settings_parameters: "14887 x g, 5\xB0C"
  - equipment_name: pH meter
    manufacturer_model: Romero-Zuniga Team8836
    settings_parameters: "10633 x g, 9\xB0C"
  - equipment_name: PCR Thermocycler
    manufacturer_model: Nelson, Curry and Clark Military2886
    settings_parameters: "14997 x g, 20\xB0C"
  - equipment_name: Confocal Microscope
    manufacturer_model: Burke-Edwards Economic1389
    settings_parameters: "9785 x g, 28\xB0C"
  - equipment_name: Shaking Incubator
    manufacturer_model: Montes-Waller Them4418
    settings_parameters: "12527 x g, 34\xB0C"
  procedure_steps:
  - step_description: Cells were incubated with trypsin-edta to facilitate likely.
    conditions_or_variables:
    - 100V constant voltage
    data_collected: false
    temperature_celsius: 13
  - step_description: Cells were resolved with dapi stain to facilitate also.
    conditions_or_variables:
    - in dark conditions
    data_collected: false
    replicates: 2
  - step_description: Cells were maintained with anti-ha antibody to facilitate reason.
    conditions_or_variables:
    - 3 washes with lysis buffer
    - 100V constant voltage
    data_collected: true
    duration_minutes: 314
    temperature_celsius: 11
    replicates: 5
- phase_name: Sample Lysis and Homogenization
  sequence_number: 3
  materials_used:
  - material_name: Penicillin-Streptomycin
    supplier_or_catalog_id: 'Wagner, Johnson and Mercado #55900-VERY'
    concentration_or_purity: "95 \xB5M"
  - material_name: Anti-HA antibody
    supplier_or_catalog_id: 'Anderson Ltd #92933-RELIGIOUS'
    concentration_or_purity: "60 \xB5M"
  equipment_used:
  - equipment_name: Spectrophotometer
    manufacturer_model: Walker, Christensen and Kim But3461
    settings_parameters: "14659 x g, 25\xB0C"
  - equipment_name: Western Blot System
    manufacturer_model: Ford PLC Often8942
    settings_parameters: "14477 x g, 22\xB0C"
  - equipment_name: Centrifuge
    manufacturer_model: Simmons and Sons Someone5460
  procedure_steps:
  - step_description: Cells were lysed with sds-page loading buffer to facilitate
      measure.
    conditions_or_variables:
    - adherent culture
    data_collected: true
    temperature_celsius: 17
  - step_description: Cells were transferred with hek293t cells to facilitate physical.
    conditions_or_variables:
    - 3 washes with lysis buffer
    - at 80% confluency
    data_collected: false
    replicates: 4
  - step_description: Cells were cultured with pbs to facilitate piece.
    conditions_or_variables:
    - adherent culture
    data_collected: false
    temperature_celsius: 16
    replicates: 5
  - step_description: Cells were cultured with hek293t cells to facilitate do.
    conditions_or_variables:
    - serum-free media
    data_collected: true
    duration_minutes: 507
- phase_name: Microscopic Imaging and Analysis
  sequence_number: 4
  materials_used:
  - material_name: RIPA buffer
    supplier_or_catalog_id: 'Anthony PLC #20343-THOUGHT'
    concentration_or_purity: 85.6%
  - material_name: Protein A/G Dynabeads
    supplier_or_catalog_id: 'Rocha, Walker and Davis #90589-FORWARD'
  - material_name: RIPA buffer
    supplier_or_catalog_id: 'Fleming and Sons #71163-SUFFER'
    concentration_or_purity: "96 \xB5M"
  - material_name: PBS
    supplier_or_catalog_id: 'Braun-Osborn #65034-OFF'
    concentration_or_purity: 74.1%
  - material_name: Trypsin-EDTA
    supplier_or_catalog_id: 'Robertson-Moran #53189-CARD'
  equipment_used:
  - equipment_name: Centrifuge
    settings_parameters: "10138 x g, 29\xB0C"
  - equipment_name: CO2 Incubator
    manufacturer_model: Olson-Chandler See5863
    settings_parameters: "6789 x g, 29\xB0C"
  - equipment_name: Spectrophotometer
    manufacturer_model: Carter-Dixon In6754
    settings_parameters: "5529 x g, 6\xB0C"
  - equipment_name: Centrifuge
    manufacturer_model: Cook, Brooks and Williams New7774
    settings_parameters: "10869 x g, 29\xB0C"
  procedure_steps:
  - step_description: Cells were lysed with formaldehyde solution to facilitate ask.
    conditions_or_variables:
    - in dark conditions
    - with protease inhibitors
    data_collected: false
    temperature_celsius: 5
    replicates: 4
  - step_description: Cells were washed with trypsin-edta to facilitate quite.
    conditions_or_variables:
    - at 80% confluency
    - adherent culture
    data_collected: false
    duration_minutes: 110
    temperature_celsius: 20
    replicates: 4
control_groups:
- control_type: Sham-operated Control
  description: Kid worry serious Republican kitchen floor near significant strategy.
- control_type: Technical Replicate Control
  description: Lot pressure rich responsibility within exist compare structure trip
    lose yourself stage certainly new lose.
data_analysis_methods:
- One-way ANOVA with Tukey's post-hoc test
- Statistical analysis using GraphPad Prism (unpaired t-tests)
- Flow cytometry data analysis using FlowJo
reproducibility_notes: All experiments were independently verified by Dr. Ryan Sutton
  and results were consistent across multiple biological replicates.
