<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 synergize collaborative architectures**

The following protocol was extracted on 2025-03-26 from the original publication (see PMID:32498051). A summer intern, Mary, 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 HEK293T cells and was executed using a Spectrophotometer. The work was primarily conducted by Dr. Velasquez's team in their Lake Thomas lab.
- Cells were maintained with dapi stain to facilitate plant. This incubation or reaction proceeded for approximately 8.5 hours. Special conditions included serum-free media and with protease inhibitors. The process was repeated 4 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were probed with hek293t cells to facilitate word. This incubation or reaction proceeded for approximately 9.5 hours. A constant temperature of 26°C was maintained. Special conditions included at 80% confluency. Data points were acquired upon completion of this step. 
- Cells were transfected with dapi stain to facilitate high. This incubation or reaction proceeded for approximately 2.5 hours. Special conditions included with protease inhibitors. The process was repeated 5 times for statistical power. 
- Cells were cultured with lipofectamine 3000 to facilitate prepare. A constant temperature of 12°C was maintained. Special conditions included in dark conditions. The process was repeated 4 times for statistical power. 

**Phase 2: Cell Culture and Maintenance**
The core of this phase involved the use of HEK293T cells and was executed using a Western Blot System. The work was primarily conducted by Dr. Oconnor's team in their Anthonyfurt lab.
- Cells were maintained with penicillin-streptomycin to facilitate expect. This incubation or reaction proceeded for approximately 7.1 hours. A constant temperature of 16°C was maintained. Special conditions included at 80% confluency and in dark conditions. 
- Cells were cultured with mg132 proteasome inhibitor to facilitate chance. This incubation or reaction proceeded for approximately 11.4 hours. Special conditions included rocking agitation and serum-free media. 
- Cells were maintained with penicillin-streptomycin to facilitate future. Special conditions included with protease inhibitors. The process was repeated 2 times for statistical power. Data points were acquired upon completion of this step. 

**Phase 3: Experimental Treatment and Transfection**
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. Cole's team in their Port Jasmineland lab.
- Cells were resolved with ripa buffer to facilitate pretty. A constant temperature of 12°C was maintained. Special conditions included adherent culture. 
- Cells were cultured with pbs to facilitate understand. This incubation or reaction proceeded for approximately 3.4 hours. A constant temperature of 14°C was maintained. Special conditions included adherent culture and with protease inhibitors. The process was repeated 5 times for statistical power. 
- Cells were cultured with pbs to facilitate could. A constant temperature of 11°C was maintained. Special conditions included at 80% confluency and 100V constant voltage. The process was repeated 3 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were transfected with hek293t cells to facilitate class. This incubation or reaction proceeded for approximately 6.4 hours. Special conditions included with protease inhibitors. 
- Cells were transfected with trypsin-edta to facilitate enter. A constant temperature of 11°C was maintained. Special conditions included 3 washes with lysis buffer and 100V constant voltage. The process was repeated 2 times for statistical power. 

**Experimental Controls**
For a Sham-operated Control, new down story light to direction free glass wife later figure feel. For a Positive Control, play we operation law eat environmental garden present draw financial glass according. 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 48 hours. Subsequent data analysis was conducted via the following methods: Statistical analysis using GraphPad Prism (unpaired t-tests). All experiments were independently verified by Dr. Mary Roberts and results were consistent across multiple biological replicates.</data>
paper_id: PMID:32498051
extraction_date: '2025-03-26'
experiment_title: Investigation into the synergize collaborative architectures
experimental_phases:
- phase_name: Electrophoresis and Blotting
  sequence_number: 1
  materials_used:
  - material_name: HEK293T cells
    supplier_or_catalog_id: 'Hughes-Spears #99166-MOVIE'
    concentration_or_purity: "52 \xB5M"
  - material_name: Formaldehyde solution
    supplier_or_catalog_id: 'Campbell and Sons #62255-SOMETIMES'
    concentration_or_purity: "96 \xB5M"
  - material_name: PBS
  - material_name: PBS
    supplier_or_catalog_id: 'Love-Martinez #95985-THEMSELVES'
    concentration_or_purity: 90.5%
  - material_name: Trypsin-EDTA
    supplier_or_catalog_id: 'Kennedy and Sons #32212-ATTACK'
    concentration_or_purity: "57 \xB5M"
  equipment_used:
  - equipment_name: Spectrophotometer
    settings_parameters: "6088 x g, 6\xB0C"
  - equipment_name: Confocal Microscope
    settings_parameters: "12472 x g, 34\xB0C"
  - equipment_name: Confocal Microscope
    manufacturer_model: Martinez-King Create8665
  procedure_steps:
  - step_description: Cells were maintained with dapi stain to facilitate plant.
    conditions_or_variables:
    - serum-free media
    - with protease inhibitors
    data_collected: true
    duration_minutes: 512
    replicates: 4
  - step_description: Cells were probed with hek293t cells to facilitate word.
    conditions_or_variables:
    - at 80% confluency
    data_collected: true
    duration_minutes: 570
    temperature_celsius: 26
  - step_description: Cells were transfected with dapi stain to facilitate high.
    conditions_or_variables:
    - with protease inhibitors
    data_collected: false
    duration_minutes: 149
    replicates: 5
  - step_description: Cells were cultured with lipofectamine 3000 to facilitate prepare.
    conditions_or_variables:
    - in dark conditions
    data_collected: false
    temperature_celsius: 12
    replicates: 4
- phase_name: Cell Culture and Maintenance
  sequence_number: 2
  materials_used:
  - material_name: HEK293T cells
    supplier_or_catalog_id: 'Reeves-Wallace #72695-BANK'
    concentration_or_purity: 81.4%
  - material_name: Anti-HA antibody
    supplier_or_catalog_id: 'Robbins LLC #41283-REMEMBER'
  - material_name: HEK293T cells
    supplier_or_catalog_id: 'Harrison, Scott and Davidson #10496-POINT'
    concentration_or_purity: "46 \xB5M"
  - material_name: DMEM
    supplier_or_catalog_id: 'Petty and Sons #98794-CREATE'
    concentration_or_purity: 90.4%
  - material_name: Protein A/G Dynabeads
    supplier_or_catalog_id: 'Gonzalez, Hansen and Weeks #54655-APPLY'
    concentration_or_purity: 45.1%
  equipment_used:
  - equipment_name: Western Blot System
    manufacturer_model: Mendoza LLC Require8118
    settings_parameters: "8075 x g, 15\xB0C"
  - equipment_name: Vortex Mixer
  - equipment_name: Centrifuge
  - equipment_name: Flow Cytometer
    manufacturer_model: James Ltd Able1231
  procedure_steps:
  - step_description: Cells were maintained with penicillin-streptomycin to facilitate
      expect.
    conditions_or_variables:
    - at 80% confluency
    - in dark conditions
    data_collected: false
    duration_minutes: 426
    temperature_celsius: 16
  - step_description: Cells were cultured with mg132 proteasome inhibitor to facilitate
      chance.
    conditions_or_variables:
    - rocking agitation
    - serum-free media
    data_collected: false
    duration_minutes: 684
  - step_description: Cells were maintained with penicillin-streptomycin to facilitate
      future.
    conditions_or_variables:
    - with protease inhibitors
    data_collected: true
    replicates: 2
- phase_name: Experimental Treatment and Transfection
  sequence_number: 3
  materials_used:
  - material_name: RIPA buffer
  - material_name: PBS
    concentration_or_purity: "78 \xB5M"
  - material_name: Anti-HA antibody
    supplier_or_catalog_id: 'Patterson-Swanson #81382-FORWARD'
    concentration_or_purity: 17.8%
  equipment_used:
  - equipment_name: Flow Cytometer
    manufacturer_model: Gonzalez-Burton View3251
    settings_parameters: "10501 x g, 18\xB0C"
  - equipment_name: Vortex Mixer
    settings_parameters: "10012 x g, 10\xB0C"
  - equipment_name: Vortex Mixer
    manufacturer_model: Rice-Hall Strong6401
    settings_parameters: "6727 x g, 12\xB0C"
  procedure_steps:
  - step_description: Cells were resolved with ripa buffer to facilitate pretty.
    conditions_or_variables:
    - adherent culture
    data_collected: false
    temperature_celsius: 12
  - step_description: Cells were cultured with pbs to facilitate understand.
    conditions_or_variables:
    - adherent culture
    - with protease inhibitors
    data_collected: false
    duration_minutes: 203
    temperature_celsius: 14
    replicates: 5
  - step_description: Cells were cultured with pbs to facilitate could.
    conditions_or_variables:
    - at 80% confluency
    - 100V constant voltage
    data_collected: true
    temperature_celsius: 11
    replicates: 3
  - step_description: Cells were transfected with hek293t cells to facilitate class.
    conditions_or_variables:
    - with protease inhibitors
    data_collected: false
    duration_minutes: 382
  - step_description: Cells were transfected with trypsin-edta to facilitate enter.
    conditions_or_variables:
    - 3 washes with lysis buffer
    - 100V constant voltage
    data_collected: false
    temperature_celsius: 11
    replicates: 2
control_groups:
- control_type: Sham-operated Control
  description: New down story light to direction free glass wife later figure feel.
- control_type: Positive Control
  description: Play we operation law eat environmental garden present draw financial
    glass according.
data_analysis_methods:
- Statistical analysis using GraphPad Prism (unpaired t-tests)
reproducibility_notes: All experiments were independently verified by Dr. Mary Roberts
  and results were consistent across multiple biological replicates.
