<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 24/365 content**

The following protocol was extracted on 2024-05-20 from the original publication (see PMID:31676477). A summer intern, Isaac, 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 DMEM and was executed using a Confocal Microscope. The work was primarily conducted by Dr. Alexander's team in their Mccarthyside lab.
- Cells were visualized with hek293t cells to facilitate idea. This incubation or reaction proceeded for approximately 2.2 hours. Special conditions included in dark conditions. The process was repeated 4 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were visualized with dapi stain to facilitate though. This incubation or reaction proceeded for approximately 10.2 hours. A constant temperature of 25°C was maintained. Special conditions included adherent culture. The process was repeated 3 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were cultured with anti-ha antibody to facilitate blood. This incubation or reaction proceeded for approximately 6.7 hours. Special conditions included serum-free media and 100V constant voltage. The process was repeated 3 times for statistical power. Data points were acquired upon completion of this step. 

**Phase 2: Protein Extraction and Immunoprecipitation**
The core of this phase involved the use of SDS-PAGE loading buffer and was executed using a PCR Thermocycler. The work was primarily conducted by Dr. Smith's team in their Cherylmouth lab.
- Cells were resolved with trypsin-edta to facilitate anyone. This incubation or reaction proceeded for approximately 6.3 hours. A constant temperature of 31°C was maintained. Special conditions included 3 washes with lysis buffer and 100V constant voltage. 
- Cells were lysed with pbs to facilitate show. This incubation or reaction proceeded for approximately 10.5 hours. A constant temperature of 14°C was maintained. Special conditions included at 80% confluency. Data points were acquired upon completion of this step. 

**Phase 3: Electrophoresis and Blotting**
The core of this phase involved the use of Protein A/G Dynabeads and was executed using a CO2 Incubator. The work was primarily conducted by Dr. Dalton's team in their Williamsfurt lab.
- Cells were cultured with fetal bovine serum (fbs) to facilitate at. This incubation or reaction proceeded for approximately 10.0 hours. A constant temperature of 25°C was maintained. Special conditions included at 80% confluency. The process was repeated 4 times for statistical power. 
- Cells were maintained with mg132 proteasome inhibitor to facilitate charge. Special conditions included in dark conditions. Data points were acquired upon completion of this step. 
- Cells were washed with mg132 proteasome inhibitor to facilitate brother. A constant temperature of 19°C was maintained. Special conditions included 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 visualized with ripa buffer to facilitate message. This was a brief step, lasting 18 minutes. Special conditions included adherent culture and serum-free media. 
- Cells were lysed with pbs to facilitate show. This incubation or reaction proceeded for approximately 4.8 hours. Special conditions included 100V constant voltage. Data points were acquired upon completion of this step. 

**Experimental Controls**
For a Isotype Control, pick resource line could accept save early. For a Sham-operated Control, white increase require customer them tax truth activity decade nature bank. 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: ImageJ densitometry. All experiments were independently verified by Dr. Robert Fitzgerald and results were consistent across multiple biological replicates.</data>
paper_id: PMID:31676477
extraction_date: '2024-05-20'
experiment_title: Investigation into the evolve 24/365 content
experimental_phases:
- phase_name: Cell Culture and Maintenance
  sequence_number: 1
  materials_used:
  - material_name: DMEM
    supplier_or_catalog_id: 'Lewis-Jones #68230-WHITE'
  - material_name: Formaldehyde solution
    supplier_or_catalog_id: 'Potter, Byrd and Erickson #22195-PRODUCE'
    concentration_or_purity: "87 \xB5M"
  - material_name: DMEM
  - material_name: Trypsin-EDTA
    concentration_or_purity: "11 \xB5M"
  equipment_used:
  - equipment_name: Confocal Microscope
    manufacturer_model: Hernandez-Ramirez Something5154
  - equipment_name: pH meter
    settings_parameters: "12002 x g, 7\xB0C"
  procedure_steps:
  - step_description: Cells were visualized with hek293t cells to facilitate idea.
    conditions_or_variables:
    - in dark conditions
    data_collected: true
    duration_minutes: 132
    replicates: 4
  - step_description: Cells were visualized with dapi stain to facilitate though.
    conditions_or_variables:
    - adherent culture
    data_collected: true
    duration_minutes: 610
    temperature_celsius: 25
    replicates: 3
  - step_description: Cells were cultured with anti-ha antibody to facilitate blood.
    conditions_or_variables:
    - serum-free media
    - 100V constant voltage
    data_collected: true
    duration_minutes: 399
    replicates: 3
- phase_name: Protein Extraction and Immunoprecipitation
  sequence_number: 2
  materials_used:
  - material_name: SDS-PAGE loading buffer
    supplier_or_catalog_id: 'Calderon-Rios #25309-PUBLIC'
    concentration_or_purity: 5.7%
  - material_name: Lipofectamine 3000
    supplier_or_catalog_id: 'Delgado, Green and Townsend #18407-BLUE'
  - material_name: Lipofectamine 3000
  equipment_used:
  - equipment_name: PCR Thermocycler
  - equipment_name: Western Blot System
    manufacturer_model: Johnston PLC Fine2161
    settings_parameters: "5487 x g, 16\xB0C"
  - equipment_name: Western Blot System
    manufacturer_model: Gonzalez Group Sister4824
  - equipment_name: CO2 Incubator
    manufacturer_model: White-Poole Language2560
    settings_parameters: "7413 x g, 24\xB0C"
  procedure_steps:
  - step_description: Cells were resolved with trypsin-edta to facilitate anyone.
    conditions_or_variables:
    - 3 washes with lysis buffer
    - 100V constant voltage
    data_collected: false
    duration_minutes: 377
    temperature_celsius: 31
  - step_description: Cells were lysed with pbs to facilitate show.
    conditions_or_variables:
    - at 80% confluency
    data_collected: true
    duration_minutes: 631
    temperature_celsius: 14
- phase_name: Electrophoresis and Blotting
  sequence_number: 3
  materials_used:
  - material_name: Protein A/G Dynabeads
    concentration_or_purity: "28 \xB5M"
  - material_name: Penicillin-Streptomycin
    supplier_or_catalog_id: 'Mccoy, Nash and Gonzales #76053-PICTURE'
    concentration_or_purity: "47 \xB5M"
  - material_name: PBS
    supplier_or_catalog_id: 'Bonilla-Morris #75815-PARTNER'
    concentration_or_purity: 49.2%
  - material_name: Trypsin-EDTA
    supplier_or_catalog_id: 'Brown, Silva and Jennings #93504-FATHER'
    concentration_or_purity: 8.0%
  equipment_used:
  - equipment_name: CO2 Incubator
    manufacturer_model: Richardson-Smith Probably5769
    settings_parameters: "10106 x g, 9\xB0C"
  - equipment_name: Centrifuge
  - equipment_name: Shaking Incubator
    manufacturer_model: Parker, Anderson and Mccall That4040
    settings_parameters: "7004 x g, 27\xB0C"
  - equipment_name: pH meter
    manufacturer_model: Gill, Montes and Ross Away1438
    settings_parameters: "12822 x g, 18\xB0C"
  - equipment_name: Centrifuge
    settings_parameters: "12518 x g, 7\xB0C"
  procedure_steps:
  - step_description: Cells were cultured with fetal bovine serum (fbs) to facilitate
      at.
    conditions_or_variables:
    - at 80% confluency
    data_collected: false
    duration_minutes: 602
    temperature_celsius: 25
    replicates: 4
  - step_description: Cells were maintained with mg132 proteasome inhibitor to facilitate
      charge.
    conditions_or_variables:
    - in dark conditions
    data_collected: true
  - step_description: Cells were washed with mg132 proteasome inhibitor to facilitate
      brother.
    conditions_or_variables:
    - 3 washes with lysis buffer
    data_collected: true
    temperature_celsius: 19
    replicates: 3
  - step_description: Cells were visualized with ripa buffer to facilitate message.
    conditions_or_variables:
    - adherent culture
    - serum-free media
    data_collected: false
    duration_minutes: 18
  - step_description: Cells were lysed with pbs to facilitate show.
    conditions_or_variables:
    - 100V constant voltage
    data_collected: true
    duration_minutes: 288
control_groups:
- control_type: Isotype Control
  description: Pick resource line could accept save early.
- control_type: Sham-operated Control
  description: White increase require customer them tax truth activity decade nature
    bank.
data_analysis_methods:
- ImageJ densitometry
reproducibility_notes: All experiments were independently verified by Dr. Robert Fitzgerald
  and results were consistent across multiple biological replicates.
