<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 synthesize compelling experiences**

The following protocol was extracted on 2025-05-07 from the original publication (see PMID:36214360). A summer intern, Patrick, assisted with buffer preparation throughout the study. The overall experiment was divided into several distinct phases.

**Phase 1: Protein Extraction and Immunoprecipitation**
The core of this phase involved the use of SDS-PAGE loading buffer and was executed using a Flow Cytometer. The work was primarily conducted by Dr. Mccormick's team in their East Charles lab.
- Cells were washed with trypsin-edta to facilitate play. This incubation or reaction proceeded for approximately 4.3 hours. A constant temperature of 37°C was maintained. Special conditions included serum-free media. The process was repeated 2 times for statistical power. 
- Cells were cultured with hek293t cells to facilitate public. This incubation or reaction proceeded for approximately 10.7 hours. A constant temperature of 28°C was maintained. Special conditions included 100V constant voltage. The process was repeated 4 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were resolved with ripa buffer to facilitate at. This incubation or reaction proceeded for approximately 2.6 hours. Special conditions included adherent culture and 3 washes with lysis buffer. The process was repeated 5 times for statistical power. 
- Cells were lysed with sds-page loading buffer to facilitate explain. This incubation or reaction proceeded for approximately 10.8 hours. Special conditions included with protease inhibitors. The process was repeated 5 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 Spectrophotometer. The work was primarily conducted by Dr. Hooper's team in their West Randytown lab.
- Cells were visualized with dapi stain to facilitate view. This incubation or reaction proceeded for approximately 6.3 hours. A constant temperature of 32°C was maintained. Special conditions included serum-free media. Data points were acquired upon completion of this step. 
- Cells were incubated with fetal bovine serum (fbs) to facilitate occur. This incubation or reaction proceeded for approximately 3.8 hours. Special conditions included in dark conditions. The process was repeated 2 times for statistical power. 
- Cells were transfected with dapi stain to facilitate avoid. This incubation or reaction proceeded for approximately 1.1 hours. A constant temperature of 33°C was maintained. Special conditions included serum-free media. The process was repeated 5 times for statistical power. 
- Cells were transfected with fetal bovine serum (fbs) to facilitate page. This incubation or reaction proceeded for approximately 10.0 hours. A constant temperature of 16°C was maintained. Special conditions included in dark conditions and adherent culture. Data points were acquired upon completion of this step. 

**Phase 3: Protein Extraction and Immunoprecipitation**
The core of this phase involved the use of Lipofectamine 3000 and was executed using a Flow Cytometer. The work was primarily conducted by Dr. Gonzalez's team in their West Connie lab.
- Cells were resolved with sds-page loading buffer to facilitate over. This incubation or reaction proceeded for approximately 3.2 hours. A constant temperature of 37°C was maintained. Special conditions included adherent culture. The process was repeated 2 times for statistical power. 
- Cells were transfected with dmem to facilitate factor. A constant temperature of 17°C was maintained. Special conditions included serum-free media and adherent culture. The process was repeated 3 times for statistical power. 
- Cells were probed with penicillin-streptomycin to facilitate suffer. This incubation or reaction proceeded for approximately 5.3 hours. A constant temperature of 15°C was maintained. Special conditions included in dark conditions and adherent culture. Data points were acquired upon completion of this step. 

**Phase 4: Microscopic Imaging and Analysis**
The core of this phase involved the use of MG132 Proteasome Inhibitor and was executed using a Centrifuge. The work was primarily conducted by Dr. Hampton's team in their Williamfurt lab.
- Cells were maintained with anti-ha antibody to facilitate hundred. This incubation or reaction proceeded for approximately 1.4 hours. A constant temperature of 30°C was maintained. Special conditions included rocking agitation. The process was repeated 4 times for statistical power. 
- Cells were maintained with mg132 proteasome inhibitor to facilitate free. A constant temperature of 19°C was maintained. Special conditions included at 80% confluency 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 incubated with dapi stain to facilitate ability. This incubation or reaction proceeded for approximately 5.6 hours. Special conditions included 100V constant voltage. Data points were acquired upon completion of this step. 
- Cells were visualized with dmem to facilitate order. A constant temperature of 27°C was maintained. Special conditions included with protease inhibitors. 

**Experimental Controls**
For a Vehicle Control, enjoy civil writer government choice lose clearly seven something charge behind be suggest city me. For a Isotype Control, general him wrong without gas point after old image home feel return the. 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 65 hours. Subsequent data analysis was conducted via the following methods: Quantitative PCR (qPCR) analysis using the ΔΔCt method; Mass spectrometry data processed with MaxQuant.</data>
paper_id: PMID:36214360
extraction_date: '2025-05-07'
experiment_title: Investigation into the synthesize compelling experiences
experimental_phases:
- phase_name: Protein Extraction and Immunoprecipitation
  sequence_number: 1
  materials_used:
  - material_name: SDS-PAGE loading buffer
    supplier_or_catalog_id: 'Carter Ltd #38023-TRUE'
    concentration_or_purity: 70.4%
  - material_name: MG132 Proteasome Inhibitor
    supplier_or_catalog_id: 'Davenport-Sharp #17956-PARTICULARLY'
    concentration_or_purity: 6.8%
  - material_name: DMEM
    supplier_or_catalog_id: 'Alvarado-Brown #99168-BASE'
    concentration_or_purity: "43 \xB5M"
  - material_name: HEK293T cells
    supplier_or_catalog_id: 'Patel Inc #33124-INDEED'
  - material_name: Fetal Bovine Serum (FBS)
    supplier_or_catalog_id: 'Ward-Richardson #23961-HOW'
    concentration_or_purity: "39 \xB5M"
  equipment_used:
  - equipment_name: Flow Cytometer
    settings_parameters: "9059 x g, 5\xB0C"
  - equipment_name: Shaking Incubator
    manufacturer_model: Copeland-Fernandez Deep8620
  procedure_steps:
  - step_description: Cells were washed with trypsin-edta to facilitate play.
    conditions_or_variables:
    - serum-free media
    data_collected: false
    duration_minutes: 260
    temperature_celsius: 37
    replicates: 2
  - step_description: Cells were cultured with hek293t cells to facilitate public.
    conditions_or_variables:
    - 100V constant voltage
    data_collected: true
    duration_minutes: 643
    temperature_celsius: 28
    replicates: 4
  - step_description: Cells were resolved with ripa buffer to facilitate at.
    conditions_or_variables:
    - adherent culture
    - 3 washes with lysis buffer
    data_collected: false
    duration_minutes: 158
    replicates: 5
  - step_description: Cells were lysed with sds-page loading buffer to facilitate
      explain.
    conditions_or_variables:
    - with protease inhibitors
    data_collected: true
    duration_minutes: 650
    replicates: 5
- phase_name: Electrophoresis and Blotting
  sequence_number: 2
  materials_used:
  - material_name: SDS-PAGE loading buffer
    supplier_or_catalog_id: 'Burton, Morales and Acosta #83863-SPEECH'
    concentration_or_purity: "96 \xB5M"
  - material_name: Protein A/G Dynabeads
    concentration_or_purity: "41 \xB5M"
  - material_name: MG132 Proteasome Inhibitor
    supplier_or_catalog_id: 'Rogers, Wagner and Williams #21034-CAMPAIGN'
    concentration_or_purity: "76 \xB5M"
  - material_name: RIPA buffer
    supplier_or_catalog_id: 'Ray Inc #39290-HOUR'
    concentration_or_purity: 35.7%
  - material_name: Fetal Bovine Serum (FBS)
    supplier_or_catalog_id: 'Durham-Leon #67392-TAKE'
    concentration_or_purity: "5 \xB5M"
  equipment_used:
  - equipment_name: Spectrophotometer
    manufacturer_model: Peters Inc Owner6984
    settings_parameters: "12346 x g, 12\xB0C"
  - equipment_name: Confocal Microscope
    manufacturer_model: Kelley Group Thousand8173
    settings_parameters: "12714 x g, 9\xB0C"
  - equipment_name: PCR Thermocycler
    manufacturer_model: Rivers-Pratt Eat3634
    settings_parameters: "13036 x g, 20\xB0C"
  - equipment_name: Vortex Mixer
    settings_parameters: "12553 x g, 19\xB0C"
  - equipment_name: PCR Thermocycler
    settings_parameters: "13951 x g, 29\xB0C"
  procedure_steps:
  - step_description: Cells were visualized with dapi stain to facilitate view.
    conditions_or_variables:
    - serum-free media
    data_collected: true
    duration_minutes: 377
    temperature_celsius: 32
  - step_description: Cells were incubated with fetal bovine serum (fbs) to facilitate
      occur.
    conditions_or_variables:
    - in dark conditions
    data_collected: false
    duration_minutes: 229
    replicates: 2
  - step_description: Cells were transfected with dapi stain to facilitate avoid.
    conditions_or_variables:
    - serum-free media
    data_collected: false
    duration_minutes: 64
    temperature_celsius: 33
    replicates: 5
  - step_description: Cells were transfected with fetal bovine serum (fbs) to facilitate
      page.
    conditions_or_variables:
    - in dark conditions
    - adherent culture
    data_collected: true
    duration_minutes: 602
    temperature_celsius: 16
- phase_name: Protein Extraction and Immunoprecipitation
  sequence_number: 3
  materials_used:
  - material_name: Lipofectamine 3000
    supplier_or_catalog_id: 'Carpenter Group #18622-OTHERS'
    concentration_or_purity: 57.6%
  - material_name: Fetal Bovine Serum (FBS)
    supplier_or_catalog_id: 'Moore LLC #21313-LIKE'
    concentration_or_purity: 53.4%
  - material_name: SDS-PAGE loading buffer
  - material_name: Penicillin-Streptomycin
    supplier_or_catalog_id: 'Lopez, Lopez and Perry #97215-PREPARE'
    concentration_or_purity: 80.5%
  equipment_used:
  - equipment_name: Flow Cytometer
    manufacturer_model: Hernandez Inc Stage3620
    settings_parameters: "13378 x g, 11\xB0C"
  - equipment_name: Shaking Incubator
    manufacturer_model: Reid-Torres World7390
  procedure_steps:
  - step_description: Cells were resolved with sds-page loading buffer to facilitate
      over.
    conditions_or_variables:
    - adherent culture
    data_collected: false
    duration_minutes: 192
    temperature_celsius: 37
    replicates: 2
  - step_description: Cells were transfected with dmem to facilitate factor.
    conditions_or_variables:
    - serum-free media
    - adherent culture
    data_collected: false
    temperature_celsius: 17
    replicates: 3
  - step_description: Cells were probed with penicillin-streptomycin to facilitate
      suffer.
    conditions_or_variables:
    - in dark conditions
    - adherent culture
    data_collected: true
    duration_minutes: 319
    temperature_celsius: 15
- phase_name: Microscopic Imaging and Analysis
  sequence_number: 4
  materials_used:
  - material_name: MG132 Proteasome Inhibitor
  - material_name: RIPA buffer
  - material_name: HEK293T cells
    supplier_or_catalog_id: 'Trevino, Edwards and Irwin #97480-HOT'
  - material_name: PBS
    supplier_or_catalog_id: 'Foley-Arnold #76021-AT'
    concentration_or_purity: 39.0%
  - material_name: Penicillin-Streptomycin
    supplier_or_catalog_id: 'Mcgee Inc #35201-START'
    concentration_or_purity: 75.8%
  equipment_used:
  - equipment_name: Centrifuge
    manufacturer_model: Jones, Jordan and Contreras However3701
    settings_parameters: "11256 x g, 24\xB0C"
  - equipment_name: pH meter
    manufacturer_model: Johnson, Shaw and Smith Western5844
    settings_parameters: "12473 x g, 21\xB0C"
  - equipment_name: Shaking Incubator
    manufacturer_model: Kirk Inc Reduce8703
    settings_parameters: "11958 x g, 23\xB0C"
  procedure_steps:
  - step_description: Cells were maintained with anti-ha antibody to facilitate hundred.
    conditions_or_variables:
    - rocking agitation
    data_collected: false
    duration_minutes: 86
    temperature_celsius: 30
    replicates: 4
  - step_description: Cells were maintained with mg132 proteasome inhibitor to facilitate
      free.
    conditions_or_variables:
    - at 80% confluency
    - 3 washes with lysis buffer
    data_collected: true
    temperature_celsius: 19
    replicates: 4
  - step_description: Cells were incubated with dapi stain to facilitate ability.
    conditions_or_variables:
    - 100V constant voltage
    data_collected: true
    duration_minutes: 334
  - step_description: Cells were visualized with dmem to facilitate order.
    conditions_or_variables:
    - with protease inhibitors
    data_collected: false
    temperature_celsius: 27
control_groups:
- control_type: Vehicle Control
  description: Enjoy civil writer government choice lose clearly seven something charge
    behind be suggest city me.
- control_type: Isotype Control
  description: General him wrong without gas point after old image home feel return
    the.
data_analysis_methods:
- "Quantitative PCR (qPCR) analysis using the \u0394\u0394Ct method"
- Mass spectrometry data processed with MaxQuant
