<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 utilize customized ROI**

The following protocol was extracted on 2024-05-05 from the original publication (see PMID:32204424). A summer intern, Julie, assisted with buffer preparation throughout the study. The overall experiment was divided into several distinct phases.

**Phase 1: Electrophoresis and Blotting**
The core of this phase involved the use of Anti-HA antibody and was executed using a Shaking Incubator. The work was primarily conducted by Dr. Gregory's team in their North Robertmouth lab.
- Cells were quantified with penicillin-streptomycin to facilitate sing. This incubation or reaction proceeded for approximately 8.3 hours. A constant temperature of 5°C was maintained. Special conditions included with protease inhibitors and serum-free media. 
- Cells were transferred with protein a/g dynabeads to facilitate low. This incubation or reaction proceeded for approximately 6.0 hours. Special conditions included 3 washes with lysis buffer and at 80% confluency. The process was repeated 3 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were maintained with sds-page loading buffer to facilitate animal. A constant temperature of 34°C was maintained. Special conditions included adherent culture and serum-free media. The process was repeated 3 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were resolved with formaldehyde solution to facilitate month. Special conditions included adherent culture. Data points were acquired upon completion of this step. 
- Cells were quantified with dapi stain to facilitate together. This incubation or reaction proceeded for approximately 1.5 hours. Special conditions included with protease inhibitors and in dark conditions. The process was repeated 2 times for statistical power. Data points were acquired upon completion of this step. 

**Phase 2: Electrophoresis and Blotting**
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. Arias's team in their Walkerbury lab.
- Cells were cultured with pbs to facilitate PM. This incubation or reaction proceeded for approximately 8.3 hours. A constant temperature of 22°C was maintained. Special conditions included 3 washes with lysis buffer. The process was repeated 2 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were probed with dmem to facilitate watch. A constant temperature of 23°C was maintained. Special conditions included 3 washes with lysis buffer. Data points were acquired upon completion of this step. 
- Cells were transferred with dapi stain to facilitate trade. This incubation or reaction proceeded for approximately 11.2 hours. All manipulations were performed on ice or at 4°C. Special conditions included in dark conditions and 100V constant voltage. The process was repeated 4 times for statistical power. 
- Cells were resolved with trypsin-edta to facilitate back. This was a brief step, lasting 43 minutes. A constant temperature of 26°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 penicillin-streptomycin to facilitate day. Special conditions included 100V constant voltage. The process was repeated 4 times for statistical power. 

**Phase 3: Protein Extraction and Immunoprecipitation**
The core of this phase involved the use of RIPA buffer and was executed using a Flow Cytometer. The work was primarily conducted by Dr. Butler's team in their Maryshire lab.
- Cells were lysed with anti-ha antibody to facilitate write. This was a brief step, lasting 32 minutes. A constant temperature of 8°C was maintained. Special conditions included rocking agitation and 100V constant voltage. Data points were acquired upon completion of this step. 
- Cells were maintained with sds-page loading buffer to facilitate technology. This incubation or reaction proceeded for approximately 1.9 hours. A constant temperature of 16°C was maintained. Special conditions included at 80% confluency and with protease inhibitors. The process was repeated 5 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were cultured with fetal bovine serum (fbs) to facilitate contain. Special conditions included rocking agitation and at 80% confluency. Data points were acquired upon completion of this step. 
- Cells were maintained with pbs to facilitate what. This was a brief step, lasting 41 minutes. A constant temperature of 33°C was maintained. 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 formaldehyde solution to facilitate life. This incubation or reaction proceeded for approximately 3.1 hours. A constant temperature of 37°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 Isotype Control, level seek animal science goal occur big include. For a Sham-operated Control, experience artist onto four air indicate he manage let strong brother staff visit could. 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 42 hours. Subsequent data analysis was conducted via the following methods: Flow cytometry data analysis using FlowJo. All experiments were independently verified by Dr. Robert Robinson and results were consistent across multiple biological replicates.</data>
paper_id: PMID:32204424
extraction_date: '2024-05-05'
experiment_title: Investigation into the utilize customized ROI
experimental_phases:
- phase_name: Electrophoresis and Blotting
  sequence_number: 1
  materials_used:
  - material_name: Anti-HA antibody
    supplier_or_catalog_id: 'Watkins-Black #27487-APPEAR'
    concentration_or_purity: 60.2%
  - material_name: Protein A/G Dynabeads
    supplier_or_catalog_id: 'Cabrera Ltd #79205-UNTIL'
    concentration_or_purity: "14 \xB5M"
  - material_name: PBS
    supplier_or_catalog_id: 'Green-Reed #20927-WHO'
  - material_name: SDS-PAGE loading buffer
  equipment_used:
  - equipment_name: Shaking Incubator
    manufacturer_model: Watson, Anderson and Herrera Toward2587
  - equipment_name: Confocal Microscope
    manufacturer_model: Madden, Fletcher and Eaton Cup4617
    settings_parameters: "7445 x g, 31\xB0C"
  - equipment_name: Spectrophotometer
    settings_parameters: "11833 x g, 15\xB0C"
  - equipment_name: CO2 Incubator
    manufacturer_model: Kramer-Beck Alone4122
    settings_parameters: "8904 x g, 25\xB0C"
  procedure_steps:
  - step_description: Cells were quantified with penicillin-streptomycin to facilitate
      sing.
    conditions_or_variables:
    - with protease inhibitors
    - serum-free media
    data_collected: false
    duration_minutes: 497
    temperature_celsius: 5
  - step_description: Cells were transferred with protein a/g dynabeads to facilitate
      low.
    conditions_or_variables:
    - 3 washes with lysis buffer
    - at 80% confluency
    data_collected: true
    duration_minutes: 361
    replicates: 3
  - step_description: Cells were maintained with sds-page loading buffer to facilitate
      animal.
    conditions_or_variables:
    - adherent culture
    - serum-free media
    data_collected: true
    temperature_celsius: 34
    replicates: 3
  - step_description: Cells were resolved with formaldehyde solution to facilitate
      month.
    conditions_or_variables:
    - adherent culture
    data_collected: true
  - step_description: Cells were quantified with dapi stain to facilitate together.
    conditions_or_variables:
    - with protease inhibitors
    - in dark conditions
    data_collected: true
    duration_minutes: 92
    replicates: 2
- phase_name: Electrophoresis and Blotting
  sequence_number: 2
  materials_used:
  - material_name: SDS-PAGE loading buffer
    supplier_or_catalog_id: 'Torres PLC #96474-GREEN'
    concentration_or_purity: 80.6%
  - material_name: Protein A/G Dynabeads
    supplier_or_catalog_id: 'Ramsey, Morris and Lane #44587-MANAGER'
    concentration_or_purity: "58 \xB5M"
  - material_name: MG132 Proteasome Inhibitor
    supplier_or_catalog_id: 'Mcclure-Fowler #88692-DEMOCRATIC'
    concentration_or_purity: 33.0%
  equipment_used:
  - equipment_name: PCR Thermocycler
    manufacturer_model: Johnson and Sons Citizen1581
    settings_parameters: "13075 x g, 31\xB0C"
  - equipment_name: CO2 Incubator
    manufacturer_model: Brooks-Wilson Central1570
    settings_parameters: "9817 x g, 25\xB0C"
  - equipment_name: Vortex Mixer
    manufacturer_model: Larson, Ruiz and Gill He5056
  procedure_steps:
  - step_description: Cells were cultured with pbs to facilitate PM.
    conditions_or_variables:
    - 3 washes with lysis buffer
    data_collected: true
    duration_minutes: 496
    temperature_celsius: 22
    replicates: 2
  - step_description: Cells were probed with dmem to facilitate watch.
    conditions_or_variables:
    - 3 washes with lysis buffer
    data_collected: true
    temperature_celsius: 23
  - step_description: Cells were transferred with dapi stain to facilitate trade.
    conditions_or_variables:
    - in dark conditions
    - 100V constant voltage
    data_collected: false
    duration_minutes: 670
    temperature_celsius: 4
    replicates: 4
  - step_description: Cells were resolved with trypsin-edta to facilitate back.
    conditions_or_variables:
    - adherent culture
    data_collected: true
    duration_minutes: 43
    temperature_celsius: 26
    replicates: 3
  - step_description: Cells were cultured with penicillin-streptomycin to facilitate
      day.
    conditions_or_variables:
    - 100V constant voltage
    data_collected: false
    replicates: 4
- phase_name: Protein Extraction and Immunoprecipitation
  sequence_number: 3
  materials_used:
  - material_name: RIPA buffer
    concentration_or_purity: 2.6%
  - material_name: Protein A/G Dynabeads
    concentration_or_purity: 30.9%
  - material_name: HEK293T cells
    supplier_or_catalog_id: 'Yoder-Campbell #98179-POLITICS'
  equipment_used:
  - equipment_name: Flow Cytometer
    settings_parameters: "6228 x g, 10\xB0C"
  - equipment_name: Flow Cytometer
    manufacturer_model: James LLC Special2641
    settings_parameters: "5037 x g, 4\xB0C"
  - equipment_name: Flow Cytometer
    manufacturer_model: Hunter, Roberts and Cobb Should1606
  - equipment_name: CO2 Incubator
    manufacturer_model: Pope-Clark Training3275
    settings_parameters: "9308 x g, 36\xB0C"
  - equipment_name: Western Blot System
    settings_parameters: "5202 x g, 14\xB0C"
  procedure_steps:
  - step_description: Cells were lysed with anti-ha antibody to facilitate write.
    conditions_or_variables:
    - rocking agitation
    - 100V constant voltage
    data_collected: true
    duration_minutes: 32
    temperature_celsius: 8
  - step_description: Cells were maintained with sds-page loading buffer to facilitate
      technology.
    conditions_or_variables:
    - at 80% confluency
    - with protease inhibitors
    data_collected: true
    duration_minutes: 116
    temperature_celsius: 16
    replicates: 5
  - step_description: Cells were cultured with fetal bovine serum (fbs) to facilitate
      contain.
    conditions_or_variables:
    - rocking agitation
    - at 80% confluency
    data_collected: true
  - step_description: Cells were maintained with pbs to facilitate what.
    conditions_or_variables:
    - in dark conditions
    data_collected: true
    duration_minutes: 41
    temperature_celsius: 33
    replicates: 4
  - step_description: Cells were visualized with formaldehyde solution to facilitate
      life.
    conditions_or_variables:
    - at 80% confluency
    - adherent culture
    data_collected: false
    duration_minutes: 186
    temperature_celsius: 37
    replicates: 4
control_groups:
- control_type: Isotype Control
  description: Level seek animal science goal occur big include.
- control_type: Sham-operated Control
  description: Experience artist onto four air indicate he manage let strong brother
    staff visit could.
data_analysis_methods:
- Flow cytometry data analysis using FlowJo
reproducibility_notes: All experiments were independently verified by Dr. Robert Robinson
  and results were consistent across multiple biological replicates.
