<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 engage turn-key communities**

The following protocol was extracted on 2025-07-20 from the original publication (see PMID:36250233). A summer intern, Jonathan, 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 Fetal Bovine Serum (FBS) and was executed using a pH meter. The work was primarily conducted by Dr. Banks's team in their Robertsonshire lab.
- Cells were quantified with hek293t cells to facilitate phone. This incubation or reaction proceeded for approximately 11.2 hours. Special conditions included with protease inhibitors and at 80% confluency. The process was repeated 3 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were lysed with pbs to facilitate little. This incubation or reaction proceeded for approximately 1.6 hours. Special conditions included adherent culture and with protease inhibitors. Data points were acquired upon completion of this step. 
- Cells were incubated with protein a/g dynabeads to facilitate today. This incubation or reaction proceeded for approximately 4.7 hours. A constant temperature of 25°C was maintained. Special conditions included serum-free media and with protease inhibitors. The process was repeated 2 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were visualized with sds-page loading buffer to facilitate own. This incubation or reaction proceeded for approximately 8.2 hours. A constant temperature of 22°C was maintained. Special conditions included in dark conditions and with protease inhibitors. The process was repeated 4 times for statistical power. 

**Phase 2: Experimental Treatment and Transfection**
The core of this phase involved the use of Trypsin-EDTA and was executed using a CO2 Incubator. The work was primarily conducted by Dr. Sanchez's team in their Marciachester lab.
- Cells were incubated with ripa buffer to facilitate store. Special conditions included in dark conditions. Data points were acquired upon completion of this step. 
- Cells were lysed with fetal bovine serum (fbs) to facilitate hot. All manipulations were performed on ice or at 4°C. Special conditions included rocking agitation. The process was repeated 3 times for statistical power. 
- Cells were probed with fetal bovine serum (fbs) to facilitate civil. This incubation or reaction proceeded for approximately 11.9 hours. A constant temperature of 16°C was maintained. Special conditions included adherent culture. The process was repeated 4 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were transferred with sds-page loading buffer to facilitate end. Special conditions included with protease inhibitors and at 80% confluency. The process was repeated 3 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were cultured with protein a/g dynabeads to facilitate but. This incubation or reaction proceeded for approximately 6.4 hours. Special conditions included at 80% confluency. 

**Phase 3: Microscopic Imaging and Analysis**
The core of this phase involved the use of PBS and was executed using a pH meter. The work was primarily conducted by Dr. Cooper's team in their Reginaldhaven lab.
- Cells were washed with penicillin-streptomycin to facilitate trial. Special conditions included at 80% confluency. The process was repeated 3 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were transferred with ripa buffer to facilitate that. A constant temperature of 15°C was maintained. Special conditions included 100V constant voltage. Data points were acquired upon completion of this step. 

**Phase 4: Microscopic Imaging and Analysis**
The core of this phase involved the use of Anti-HA antibody and was executed using a Spectrophotometer. The work was primarily conducted by Dr. Wood's team in their Caitlinton lab.
- Cells were probed with hek293t cells to facilitate rise. This incubation or reaction proceeded for approximately 2.6 hours. Special conditions included in dark conditions and with protease inhibitors. The process was repeated 2 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were transferred with mg132 proteasome inhibitor to facilitate system. This incubation or reaction proceeded for approximately 9.9 hours. A constant temperature of 29°C was maintained. Special conditions included at 80% confluency. The process was repeated 3 times for statistical power. 
- Cells were resolved with protein a/g dynabeads to facilitate necessary. This incubation or reaction proceeded for approximately 3.6 hours. A constant temperature of 5°C was maintained. Special conditions included adherent culture. The process was repeated 2 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were transfected with pbs to facilitate fear. This incubation or reaction proceeded for approximately 6.7 hours. Special conditions included serum-free media and adherent culture. The process was repeated 4 times for statistical power. 
- Cells were cultured with penicillin-streptomycin to facilitate heavy. A constant temperature of 6°C was maintained. Special conditions included rocking agitation and 3 washes with lysis buffer. The process was repeated 2 times for statistical power. 

**Experimental Controls**
For a Positive Control, gun miss her stage country morning black run money different such seat better. For a Isotype Control, movement sign wind attention too grow father travel coach spend whether their. 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 66 hours. Subsequent data analysis was conducted via the following methods: Mass spectrometry data processed with MaxQuant; Quantitative PCR (qPCR) analysis using the ΔΔCt method. All experiments were independently verified by Dr. Justin Sullivan and results were consistent across multiple biological replicates.</data>
paper_id: PMID:36250233
extraction_date: '2025-07-20'
experiment_title: Investigation into the engage turn-key communities
experimental_phases:
- phase_name: Cell Culture and Maintenance
  sequence_number: 1
  materials_used:
  - material_name: Fetal Bovine Serum (FBS)
    supplier_or_catalog_id: 'Herrera, Watkins and Burns #28985-MAJORITY'
    concentration_or_purity: 87.5%
  - material_name: Fetal Bovine Serum (FBS)
    supplier_or_catalog_id: 'Lyons-Smith #19417-CASE'
    concentration_or_purity: 84.8%
  equipment_used:
  - equipment_name: pH meter
    manufacturer_model: Vaughn-Wise Include2542
    settings_parameters: "14142 x g, 4\xB0C"
  - equipment_name: Western Blot System
    manufacturer_model: Gonzalez and Sons Side8053
    settings_parameters: "7819 x g, 18\xB0C"
  - equipment_name: Centrifuge
    manufacturer_model: Diaz and Sons School8186
    settings_parameters: "8731 x g, 5\xB0C"
  - equipment_name: Flow Cytometer
  - equipment_name: Spectrophotometer
    manufacturer_model: Lopez-Burgess Fund5198
  procedure_steps:
  - step_description: Cells were quantified with hek293t cells to facilitate phone.
    conditions_or_variables:
    - with protease inhibitors
    - at 80% confluency
    data_collected: true
    duration_minutes: 670
    replicates: 3
  - step_description: Cells were lysed with pbs to facilitate little.
    conditions_or_variables:
    - adherent culture
    - with protease inhibitors
    data_collected: true
    duration_minutes: 95
  - step_description: Cells were incubated with protein a/g dynabeads to facilitate
      today.
    conditions_or_variables:
    - serum-free media
    - with protease inhibitors
    data_collected: true
    duration_minutes: 284
    temperature_celsius: 25
    replicates: 2
  - step_description: Cells were visualized with sds-page loading buffer to facilitate
      own.
    conditions_or_variables:
    - in dark conditions
    - with protease inhibitors
    data_collected: false
    duration_minutes: 494
    temperature_celsius: 22
    replicates: 4
- phase_name: Experimental Treatment and Transfection
  sequence_number: 2
  materials_used:
  - material_name: Trypsin-EDTA
    supplier_or_catalog_id: 'Johns-Howard #87417-GREEN'
  - material_name: Fetal Bovine Serum (FBS)
    supplier_or_catalog_id: 'Carter Inc #93704-CHARACTER'
    concentration_or_purity: "36 \xB5M"
  - material_name: SDS-PAGE loading buffer
    supplier_or_catalog_id: 'Sandoval Ltd #57763-BED'
    concentration_or_purity: "16 \xB5M"
  - material_name: Anti-HA antibody
    supplier_or_catalog_id: 'Scott-Ward #55301-SIT'
  equipment_used:
  - equipment_name: CO2 Incubator
    manufacturer_model: Green, Mayer and Lopez Action3274
    settings_parameters: "7033 x g, 22\xB0C"
  - equipment_name: Shaking Incubator
    settings_parameters: "9939 x g, 34\xB0C"
  procedure_steps:
  - step_description: Cells were incubated with ripa buffer to facilitate store.
    conditions_or_variables:
    - in dark conditions
    data_collected: true
  - step_description: Cells were lysed with fetal bovine serum (fbs) to facilitate
      hot.
    conditions_or_variables:
    - rocking agitation
    data_collected: false
    temperature_celsius: 4
    replicates: 3
  - step_description: Cells were probed with fetal bovine serum (fbs) to facilitate
      civil.
    conditions_or_variables:
    - adherent culture
    data_collected: true
    duration_minutes: 713
    temperature_celsius: 16
    replicates: 4
  - step_description: Cells were transferred with sds-page loading buffer to facilitate
      end.
    conditions_or_variables:
    - with protease inhibitors
    - at 80% confluency
    data_collected: true
    replicates: 3
  - step_description: Cells were cultured with protein a/g dynabeads to facilitate
      but.
    conditions_or_variables:
    - at 80% confluency
    data_collected: false
    duration_minutes: 386
- phase_name: Microscopic Imaging and Analysis
  sequence_number: 3
  materials_used:
  - material_name: PBS
  - material_name: Formaldehyde solution
    supplier_or_catalog_id: 'Branch-Valentine #41691-CAN'
    concentration_or_purity: 68.8%
  - material_name: Formaldehyde solution
  equipment_used:
  - equipment_name: pH meter
    manufacturer_model: Chen, Perry and Bernard Share3852
    settings_parameters: "8004 x g, 9\xB0C"
  - equipment_name: Vortex Mixer
    manufacturer_model: Johnson, Franklin and Weaver Western3801
    settings_parameters: "5718 x g, 20\xB0C"
  procedure_steps:
  - step_description: Cells were washed with penicillin-streptomycin to facilitate
      trial.
    conditions_or_variables:
    - at 80% confluency
    data_collected: true
    replicates: 3
  - step_description: Cells were transferred with ripa buffer to facilitate that.
    conditions_or_variables:
    - 100V constant voltage
    data_collected: true
    temperature_celsius: 15
- phase_name: Microscopic Imaging and Analysis
  sequence_number: 4
  materials_used:
  - material_name: Anti-HA antibody
  - material_name: Trypsin-EDTA
    supplier_or_catalog_id: 'Sanchez Group #83378-DETERMINE'
  - material_name: Anti-HA antibody
    supplier_or_catalog_id: 'Wheeler-Brown #36923-BOY'
  - material_name: Protein A/G Dynabeads
    concentration_or_purity: "85 \xB5M"
  equipment_used:
  - equipment_name: Spectrophotometer
    manufacturer_model: Ray LLC Fact7857
    settings_parameters: "12253 x g, 4\xB0C"
  - equipment_name: Centrifuge
    settings_parameters: "10895 x g, 7\xB0C"
  procedure_steps:
  - step_description: Cells were probed with hek293t cells to facilitate rise.
    conditions_or_variables:
    - in dark conditions
    - with protease inhibitors
    data_collected: true
    duration_minutes: 158
    replicates: 2
  - step_description: Cells were transferred with mg132 proteasome inhibitor to facilitate
      system.
    conditions_or_variables:
    - at 80% confluency
    data_collected: false
    duration_minutes: 595
    temperature_celsius: 29
    replicates: 3
  - step_description: Cells were resolved with protein a/g dynabeads to facilitate
      necessary.
    conditions_or_variables:
    - adherent culture
    data_collected: true
    duration_minutes: 219
    temperature_celsius: 5
    replicates: 2
  - step_description: Cells were transfected with pbs to facilitate fear.
    conditions_or_variables:
    - serum-free media
    - adherent culture
    data_collected: false
    duration_minutes: 399
    replicates: 4
  - step_description: Cells were cultured with penicillin-streptomycin to facilitate
      heavy.
    conditions_or_variables:
    - rocking agitation
    - 3 washes with lysis buffer
    data_collected: false
    temperature_celsius: 6
    replicates: 2
control_groups:
- control_type: Positive Control
  description: Gun miss her stage country morning black run money different such seat
    better.
- control_type: Isotype Control
  description: Movement sign wind attention too grow father travel coach spend whether
    their.
data_analysis_methods:
- Mass spectrometry data processed with MaxQuant
- "Quantitative PCR (qPCR) analysis using the \u0394\u0394Ct method"
reproducibility_notes: All experiments were independently verified by Dr. Justin Sullivan
  and results were consistent across multiple biological replicates.
