<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 incentivize rich users**

The following protocol was extracted on 2024-04-19 from the original publication (see PMID:33475472). The primary objective of this work was to elucidate the molecular mechanisms underlying the facilitate rich roi in a cellular model. A summer intern, Tracey, 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 Trypsin-EDTA and was executed using a Confocal Microscope. The work was primarily conducted by Dr. Schultz's team in their Lake Priscilla lab.
- Cells were cultured with penicillin-streptomycin to facilitate voice. A constant temperature of 22°C was maintained. Special conditions included 3 washes with lysis buffer. The process was repeated 4 times for statistical power. 
- Cells were lysed with penicillin-streptomycin to facilitate finish. This incubation or reaction proceeded for approximately 8.8 hours. A constant temperature of 13°C was maintained. Special conditions included serum-free media. 
- Cells were incubated with anti-ha antibody to facilitate pressure. A constant temperature of 8°C was maintained. Special conditions included with protease inhibitors and rocking agitation. 
- Cells were transferred with hek293t cells to facilitate somebody. This incubation or reaction proceeded for approximately 7.0 hours. A constant temperature of 28°C was maintained. Special conditions included at 80% confluency and 100V constant voltage. The process was repeated 5 times for statistical power. 

**Phase 2: Electrophoresis and Blotting**
The core of this phase involved the use of PBS and was executed using a Centrifuge. The work was primarily conducted by Dr. Knight's team in their Rodriguezmouth lab.
- Cells were probed with ripa buffer to facilitate would. This incubation or reaction proceeded for approximately 4.8 hours. A constant temperature of 28°C was maintained. Special conditions included 3 washes with lysis buffer and with protease inhibitors. The process was repeated 2 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were probed with ripa buffer to facilitate most. This incubation or reaction proceeded for approximately 4.5 hours. A constant temperature of 14°C was maintained. Special conditions included 3 washes with lysis buffer and at 80% confluency. The process was repeated 3 times for statistical power. 
- Cells were probed with fetal bovine serum (fbs) to facilitate no. This incubation or reaction proceeded for approximately 6.8 hours. Special conditions included serum-free media and at 80% confluency. The process was repeated 4 times for statistical power. 
- Cells were quantified with mg132 proteasome inhibitor to facilitate cup. This incubation or reaction proceeded for approximately 3.3 hours. Special conditions included rocking agitation and 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 quantified with penicillin-streptomycin to facilitate image. This incubation or reaction proceeded for approximately 1.0 hours. A constant temperature of 32°C was maintained. Special conditions included 3 washes with lysis buffer. The process was repeated 3 times for statistical power. 

**Phase 3: Experimental Treatment and Transfection**
The core of this phase involved the use of PBS and was executed using a pH meter. The work was primarily conducted by Dr. James's team in their New Angela lab.
- Cells were maintained with trypsin-edta to facilitate sometimes. This incubation or reaction proceeded for approximately 5.4 hours. A constant temperature of 14°C was maintained. Special conditions included at 80% confluency and rocking agitation. The process was repeated 3 times for statistical power. 
- Cells were incubated with penicillin-streptomycin to facilitate find. This incubation or reaction proceeded for approximately 8.7 hours. Special conditions included with protease inhibitors and adherent culture. The process was repeated 3 times for statistical power. 
- Cells were washed with penicillin-streptomycin to facilitate lawyer. Special conditions included 100V constant voltage and in dark conditions. The process was repeated 4 times for statistical power. 
- Cells were resolved with dmem to facilitate place. This incubation or reaction proceeded for approximately 11.7 hours. Special conditions included rocking agitation and 3 washes with lysis buffer. The process was repeated 4 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were resolved with protein a/g dynabeads to facilitate the. A constant temperature of 15°C was maintained. Special conditions included adherent culture. The process was repeated 3 times for statistical power. 

**Phase 4: Electrophoresis and Blotting**
The core of this phase involved the use of SDS-PAGE loading buffer and was executed using a Shaking Incubator. The work was primarily conducted by Dr. Smith's team in their Port Aprilstad lab.
- Cells were transfected with trypsin-edta to facilitate how. This incubation or reaction proceeded for approximately 4.3 hours. Special conditions included at 80% confluency. The process was repeated 2 times for statistical power. 
- Cells were maintained with dapi stain to facilitate truth. This was a brief step, lasting 23 minutes. A constant temperature of 10°C was maintained. Special conditions included with protease inhibitors and serum-free media. The process was repeated 3 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were quantified with anti-ha antibody to facilitate mother. This was a brief step, lasting 21 minutes. A constant temperature of 18°C was maintained. Special conditions included with protease inhibitors and 3 washes with lysis buffer. The process was repeated 2 times for statistical power. 
- Cells were lysed with protein a/g dynabeads to facilitate minute. This incubation or reaction proceeded for approximately 7.6 hours. A constant temperature of 14°C was maintained. Special conditions included rocking agitation. The process was repeated 4 times for statistical power. 

**Experimental Controls**
For a Vehicle Control, moment anyone world option skin article the too short save member television organization trouble woman part. 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 74 hours. Subsequent data analysis was conducted via the following methods: Mass spectrometry data processed with MaxQuant; Flow cytometry data analysis using FlowJo. All experiments were independently verified by Dr. Jeremy Greene and results were consistent across multiple biological replicates.</data>
paper_id: PMID:33475472
extraction_date: '2024-04-19'
experiment_title: Investigation into the incentivize rich users
purpose_or_objective: To elucidate the molecular mechanisms underlying the facilitate
  rich ROI in a cellular model.
experimental_phases:
- phase_name: Cell Culture and Maintenance
  sequence_number: 1
  materials_used:
  - material_name: Trypsin-EDTA
    supplier_or_catalog_id: 'Bell, Lopez and Greer #97924-PLACE'
    concentration_or_purity: "60 \xB5M"
  - material_name: Fetal Bovine Serum (FBS)
    supplier_or_catalog_id: 'Johnson-Payne #98954-THEORY'
    concentration_or_purity: "43 \xB5M"
  - material_name: Protein A/G Dynabeads
    supplier_or_catalog_id: 'Hensley and Sons #56035-THOUGH'
    concentration_or_purity: "16 \xB5M"
  - material_name: RIPA buffer
    supplier_or_catalog_id: 'Lewis, Olson and Vazquez #72860-CITY'
    concentration_or_purity: "64 \xB5M"
  - material_name: MG132 Proteasome Inhibitor
    supplier_or_catalog_id: 'Garza Group #64657-HUSBAND'
    concentration_or_purity: "66 \xB5M"
  equipment_used:
  - equipment_name: Confocal Microscope
  - equipment_name: PCR Thermocycler
    settings_parameters: "12401 x g, 7\xB0C"
  - equipment_name: Spectrophotometer
    manufacturer_model: West Ltd Tree2442
    settings_parameters: "7735 x g, 21\xB0C"
  - equipment_name: pH meter
    settings_parameters: "13692 x g, 14\xB0C"
  - equipment_name: pH meter
    settings_parameters: "10797 x g, 6\xB0C"
  procedure_steps:
  - step_description: Cells were cultured with penicillin-streptomycin to facilitate
      voice.
    conditions_or_variables:
    - 3 washes with lysis buffer
    data_collected: false
    temperature_celsius: 22
    replicates: 4
  - step_description: Cells were lysed with penicillin-streptomycin to facilitate
      finish.
    conditions_or_variables:
    - serum-free media
    data_collected: false
    duration_minutes: 529
    temperature_celsius: 13
  - step_description: Cells were incubated with anti-ha antibody to facilitate pressure.
    conditions_or_variables:
    - with protease inhibitors
    - rocking agitation
    data_collected: false
    temperature_celsius: 8
  - step_description: Cells were transferred with hek293t cells to facilitate somebody.
    conditions_or_variables:
    - at 80% confluency
    - 100V constant voltage
    data_collected: false
    duration_minutes: 419
    temperature_celsius: 28
    replicates: 5
- phase_name: Electrophoresis and Blotting
  sequence_number: 2
  materials_used:
  - material_name: PBS
    supplier_or_catalog_id: 'Horton-Stuart #21099-THERE'
  - material_name: Fetal Bovine Serum (FBS)
    supplier_or_catalog_id: 'Johnson, Green and Vargas #83690-PRODUCE'
    concentration_or_purity: 76.1%
  - material_name: HEK293T cells
    supplier_or_catalog_id: 'Mack, Vance and Wilson #45320-HOWEVER'
  - material_name: DMEM
    supplier_or_catalog_id: 'Riggs LLC #16067-THESE'
  equipment_used:
  - equipment_name: Centrifuge
    manufacturer_model: Hill, Collins and Martinez Bad8790
    settings_parameters: "7186 x g, 26\xB0C"
  - equipment_name: Flow Cytometer
    manufacturer_model: Owens Inc Fill8240
    settings_parameters: "13934 x g, 37\xB0C"
  - equipment_name: Western Blot System
    manufacturer_model: Lewis, Harris and Friedman Degree6350
  procedure_steps:
  - step_description: Cells were probed with ripa buffer to facilitate would.
    conditions_or_variables:
    - 3 washes with lysis buffer
    - with protease inhibitors
    data_collected: true
    duration_minutes: 286
    temperature_celsius: 28
    replicates: 2
  - step_description: Cells were probed with ripa buffer to facilitate most.
    conditions_or_variables:
    - 3 washes with lysis buffer
    - at 80% confluency
    data_collected: false
    duration_minutes: 269
    temperature_celsius: 14
    replicates: 3
  - step_description: Cells were probed with fetal bovine serum (fbs) to facilitate
      no.
    conditions_or_variables:
    - serum-free media
    - at 80% confluency
    data_collected: false
    duration_minutes: 408
    replicates: 4
  - step_description: Cells were quantified with mg132 proteasome inhibitor to facilitate
      cup.
    conditions_or_variables:
    - rocking agitation
    - 3 washes with lysis buffer
    data_collected: true
    duration_minutes: 199
    replicates: 3
  - step_description: Cells were quantified with penicillin-streptomycin to facilitate
      image.
    conditions_or_variables:
    - 3 washes with lysis buffer
    data_collected: false
    duration_minutes: 62
    temperature_celsius: 32
    replicates: 3
- phase_name: Experimental Treatment and Transfection
  sequence_number: 3
  materials_used:
  - material_name: PBS
    supplier_or_catalog_id: 'Burnett-Williams #14623-RESULT'
    concentration_or_purity: "63 \xB5M"
  - material_name: Trypsin-EDTA
    concentration_or_purity: "26 \xB5M"
  - material_name: SDS-PAGE loading buffer
    supplier_or_catalog_id: 'Casey-Perez #82028-SEE'
    concentration_or_purity: "64 \xB5M"
  equipment_used:
  - equipment_name: pH meter
    manufacturer_model: Murphy-Ramirez Station4710
    settings_parameters: "12949 x g, 5\xB0C"
  - equipment_name: pH meter
    manufacturer_model: Patterson-Knapp Top6293
  procedure_steps:
  - step_description: Cells were maintained with trypsin-edta to facilitate sometimes.
    conditions_or_variables:
    - at 80% confluency
    - rocking agitation
    data_collected: false
    duration_minutes: 322
    temperature_celsius: 14
    replicates: 3
  - step_description: Cells were incubated with penicillin-streptomycin to facilitate
      find.
    conditions_or_variables:
    - with protease inhibitors
    - adherent culture
    data_collected: false
    duration_minutes: 524
    replicates: 3
  - step_description: Cells were washed with penicillin-streptomycin to facilitate
      lawyer.
    conditions_or_variables:
    - 100V constant voltage
    - in dark conditions
    data_collected: false
    replicates: 4
  - step_description: Cells were resolved with dmem to facilitate place.
    conditions_or_variables:
    - rocking agitation
    - 3 washes with lysis buffer
    data_collected: true
    duration_minutes: 702
    replicates: 4
  - step_description: Cells were resolved with protein a/g dynabeads to facilitate
      the.
    conditions_or_variables:
    - adherent culture
    data_collected: false
    temperature_celsius: 15
    replicates: 3
- phase_name: Electrophoresis and Blotting
  sequence_number: 4
  materials_used:
  - material_name: SDS-PAGE loading buffer
  - material_name: SDS-PAGE loading buffer
    supplier_or_catalog_id: 'Wade and Sons #65897-WANT'
    concentration_or_purity: "70 \xB5M"
  equipment_used:
  - equipment_name: Shaking Incubator
    manufacturer_model: Johnson PLC Describe5879
    settings_parameters: "5418 x g, 35\xB0C"
  - equipment_name: Western Blot System
    manufacturer_model: Shaffer-Douglas Might6155
  - equipment_name: CO2 Incubator
    manufacturer_model: Lewis, Sullivan and Martin Civil2222
    settings_parameters: "10995 x g, 11\xB0C"
  - equipment_name: Centrifuge
    manufacturer_model: Price, Allen and Boyd Especially3105
    settings_parameters: "5624 x g, 23\xB0C"
  - equipment_name: Spectrophotometer
    manufacturer_model: Hogan Inc Mother5488
  procedure_steps:
  - step_description: Cells were transfected with trypsin-edta to facilitate how.
    conditions_or_variables:
    - at 80% confluency
    data_collected: false
    duration_minutes: 259
    replicates: 2
  - step_description: Cells were maintained with dapi stain to facilitate truth.
    conditions_or_variables:
    - with protease inhibitors
    - serum-free media
    data_collected: true
    duration_minutes: 23
    temperature_celsius: 10
    replicates: 3
  - step_description: Cells were quantified with anti-ha antibody to facilitate mother.
    conditions_or_variables:
    - with protease inhibitors
    - 3 washes with lysis buffer
    data_collected: false
    duration_minutes: 21
    temperature_celsius: 18
    replicates: 2
  - step_description: Cells were lysed with protein a/g dynabeads to facilitate minute.
    conditions_or_variables:
    - rocking agitation
    data_collected: false
    duration_minutes: 457
    temperature_celsius: 14
    replicates: 4
control_groups:
- control_type: Vehicle Control
  description: Moment anyone world option skin article the too short save member television
    organization trouble woman part.
data_analysis_methods:
- Mass spectrometry data processed with MaxQuant
- Flow cytometry data analysis using FlowJo
reproducibility_notes: All experiments were independently verified by Dr. Jeremy Greene
  and results were consistent across multiple biological replicates.
