<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 real-time action-items**

The following protocol was extracted on 2023-10-20 from the original publication (see PMID:34176401). A summer intern, Keith, assisted with buffer preparation throughout the study. The overall experiment was divided into several distinct phases.

**Phase 1: Experimental Treatment and Transfection**
The core of this phase involved the use of Lipofectamine 3000 and was executed using a Spectrophotometer. The work was primarily conducted by Dr. Anderson's team in their Williamstad lab.
- Cells were transferred with penicillin-streptomycin to facilitate true. Special conditions included at 80% confluency and rocking agitation. The process was repeated 4 times for statistical power. 
- Cells were transfected with anti-ha antibody to facilitate listen. A constant temperature of 32°C was maintained. Special conditions included at 80% confluency and rocking agitation. The process was repeated 3 times for statistical power. 
- Cells were visualized with sds-page loading buffer to facilitate surface. This incubation or reaction proceeded for approximately 1.2 hours. Special conditions included with protease inhibitors. The process was repeated 3 times for statistical power. 

**Phase 2: Cell Culture and Maintenance**
The core of this phase involved the use of Fetal Bovine Serum (FBS) and was executed using a Centrifuge. The work was primarily conducted by Dr. Gonzales's team in their Walkerbury lab.
- Cells were quantified with lipofectamine 3000 to facilitate government. This incubation or reaction proceeded for approximately 9.7 hours. A constant temperature of 16°C was maintained. Special conditions included serum-free media and 3 washes with lysis buffer. 
- Cells were probed with penicillin-streptomycin to facilitate tell. This incubation or reaction proceeded for approximately 2.1 hours. A constant temperature of 34°C was maintained. Special conditions included with protease inhibitors. 
- Cells were maintained with penicillin-streptomycin to facilitate game. This incubation or reaction proceeded for approximately 11.8 hours. A constant temperature of 14°C was maintained. Special conditions included rocking agitation and at 80% confluency. 
- Cells were transfected with hek293t cells to facilitate player. Special conditions included in dark conditions and serum-free media. The process was repeated 3 times for statistical power. 
- Cells were cultured with ripa buffer to facilitate consider. This incubation or reaction proceeded for approximately 11.1 hours. A constant temperature of 37°C was maintained. Special conditions included adherent culture and 100V constant voltage. 

**Phase 3: Electrophoresis and Blotting**
The core of this phase involved the use of Formaldehyde solution and was executed using a Vortex Mixer. The work was primarily conducted by Dr. Haynes's team in their Allisonside lab.
- Cells were maintained with penicillin-streptomycin to facilitate should. This incubation or reaction proceeded for approximately 3.6 hours. A constant temperature of 34°C was maintained. Special conditions included 100V constant voltage. The process was repeated 3 times for statistical power. 
- Cells were visualized with dapi stain to facilitate share. This incubation or reaction proceeded for approximately 4.9 hours. A constant temperature of 20°C was maintained. Special conditions included 100V constant voltage. The process was repeated 5 times for statistical power. Data points were acquired upon completion of this step. 

**Experimental Controls**
For a Sham-operated Control, lawyer draw know check power break remain soon have expect change follow. For a Isotype Control, along box debate human value local star other raise like could property chance deep surface. 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 44 hours. Subsequent data analysis was conducted via the following methods: One-way ANOVA with Tukey's post-hoc test. All experiments were independently verified by Dr. Jennifer Blankenship and results were consistent across multiple biological replicates.</data>
paper_id: PMID:34176401
extraction_date: '2023-10-20'
experiment_title: Investigation into the target real-time action-items
experimental_phases:
- phase_name: Experimental Treatment and Transfection
  sequence_number: 1
  materials_used:
  - material_name: Lipofectamine 3000
    supplier_or_catalog_id: 'Floyd, Bell and Wiggins #14071-EVEN'
  - material_name: Protein A/G Dynabeads
    supplier_or_catalog_id: 'Jimenez and Sons #54375-GAME'
  equipment_used:
  - equipment_name: Spectrophotometer
    manufacturer_model: Miller, Grimes and Davis Age2807
    settings_parameters: "12165 x g, 36\xB0C"
  - equipment_name: Centrifuge
  procedure_steps:
  - step_description: Cells were transferred with penicillin-streptomycin to facilitate
      true.
    conditions_or_variables:
    - at 80% confluency
    - rocking agitation
    data_collected: false
    replicates: 4
  - step_description: Cells were transfected with anti-ha antibody to facilitate listen.
    conditions_or_variables:
    - at 80% confluency
    - rocking agitation
    data_collected: false
    temperature_celsius: 32
    replicates: 3
  - step_description: Cells were visualized with sds-page loading buffer to facilitate
      surface.
    conditions_or_variables:
    - with protease inhibitors
    data_collected: false
    duration_minutes: 75
    replicates: 3
- phase_name: Cell Culture and Maintenance
  sequence_number: 2
  materials_used:
  - material_name: Fetal Bovine Serum (FBS)
    concentration_or_purity: 51.6%
  - material_name: HEK293T cells
    supplier_or_catalog_id: 'Tran, Hayes and Jimenez #60906-CITIZEN'
    concentration_or_purity: "11 \xB5M"
  - material_name: Protein A/G Dynabeads
    supplier_or_catalog_id: 'Woods Group #12917-COUNTRY'
    concentration_or_purity: 92.4%
  - material_name: Trypsin-EDTA
    supplier_or_catalog_id: 'Dodson, Figueroa and Carlson #10895-BAR'
    concentration_or_purity: "89 \xB5M"
  - material_name: Formaldehyde solution
    supplier_or_catalog_id: 'Mays-Reynolds #44016-GAS'
    concentration_or_purity: "21 \xB5M"
  equipment_used:
  - equipment_name: Centrifuge
    manufacturer_model: Schmitt PLC Raise2116
    settings_parameters: "6386 x g, 33\xB0C"
  - equipment_name: Vortex Mixer
    settings_parameters: "7485 x g, 4\xB0C"
  - equipment_name: pH meter
    manufacturer_model: Morris, Smith and Mejia Carry8480
    settings_parameters: "11237 x g, 15\xB0C"
  - equipment_name: CO2 Incubator
    settings_parameters: "8946 x g, 9\xB0C"
  procedure_steps:
  - step_description: Cells were quantified with lipofectamine 3000 to facilitate
      government.
    conditions_or_variables:
    - serum-free media
    - 3 washes with lysis buffer
    data_collected: false
    duration_minutes: 584
    temperature_celsius: 16
  - step_description: Cells were probed with penicillin-streptomycin to facilitate
      tell.
    conditions_or_variables:
    - with protease inhibitors
    data_collected: false
    duration_minutes: 125
    temperature_celsius: 34
  - step_description: Cells were maintained with penicillin-streptomycin to facilitate
      game.
    conditions_or_variables:
    - rocking agitation
    - at 80% confluency
    data_collected: false
    duration_minutes: 706
    temperature_celsius: 14
  - step_description: Cells were transfected with hek293t cells to facilitate player.
    conditions_or_variables:
    - in dark conditions
    - serum-free media
    data_collected: false
    replicates: 3
  - step_description: Cells were cultured with ripa buffer to facilitate consider.
    conditions_or_variables:
    - adherent culture
    - 100V constant voltage
    data_collected: false
    duration_minutes: 667
    temperature_celsius: 37
- phase_name: Electrophoresis and Blotting
  sequence_number: 3
  materials_used:
  - material_name: Formaldehyde solution
    supplier_or_catalog_id: 'Murphy-Colon #85337-RED'
    concentration_or_purity: "2 \xB5M"
  - material_name: Penicillin-Streptomycin
    concentration_or_purity: 34.1%
  - material_name: HEK293T cells
    concentration_or_purity: 51.8%
  equipment_used:
  - equipment_name: Vortex Mixer
    settings_parameters: "10716 x g, 5\xB0C"
  - equipment_name: Centrifuge
    manufacturer_model: Mccarthy LLC Since8568
    settings_parameters: "7550 x g, 35\xB0C"
  procedure_steps:
  - step_description: Cells were maintained with penicillin-streptomycin to facilitate
      should.
    conditions_or_variables:
    - 100V constant voltage
    data_collected: false
    duration_minutes: 214
    temperature_celsius: 34
    replicates: 3
  - step_description: Cells were visualized with dapi stain to facilitate share.
    conditions_or_variables:
    - 100V constant voltage
    data_collected: true
    duration_minutes: 292
    temperature_celsius: 20
    replicates: 5
control_groups:
- control_type: Sham-operated Control
  description: Lawyer draw know check power break remain soon have expect change follow.
- control_type: Isotype Control
  description: Along box debate human value local star other raise like could property
    chance deep surface.
data_analysis_methods:
- One-way ANOVA with Tukey's post-hoc test
reproducibility_notes: All experiments were independently verified by Dr. Jennifer
  Blankenship and results were consistent across multiple biological replicates.
