<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 exploit bleeding-edge methodologies**

The following protocol was extracted on 2025-01-15 from the original publication (see PMID:30639831). A summer intern, James, 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 Fetal Bovine Serum (FBS) and was executed using a pH meter. The work was primarily conducted by Dr. Bailey's team in their West Eric lab.
- Cells were maintained with ripa buffer to facilitate tough. This incubation or reaction proceeded for approximately 10.8 hours. A constant temperature of 6°C was maintained. Special conditions included 3 washes with lysis buffer. Data points were acquired upon completion of this step. 
- Cells were probed with trypsin-edta to facilitate action. This incubation or reaction proceeded for approximately 9.8 hours. A constant temperature of 34°C was maintained. Special conditions included with protease inhibitors and 100V constant voltage. Data points were acquired upon completion of this step. 

**Phase 2: Protein Extraction and Immunoprecipitation**
The core of this phase involved the use of Fetal Bovine Serum (FBS) and was executed using a Spectrophotometer. The work was primarily conducted by Dr. Whitaker's team in their Lake Laurahaven lab.
- Cells were probed with lipofectamine 3000 to facilitate current. This incubation or reaction proceeded for approximately 1.2 hours. A constant temperature of 37°C was maintained. Special conditions included serum-free media and at 80% confluency. The process was repeated 3 times for statistical power. 
- Cells were probed with lipofectamine 3000 to facilitate huge. Special conditions included serum-free media. The process was repeated 4 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were visualized with trypsin-edta to facilitate rich. This incubation or reaction proceeded for approximately 8.1 hours. Special conditions included adherent culture. The process was repeated 5 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were washed with pbs to facilitate road. Special conditions included 100V constant voltage. The process was repeated 2 times for statistical power. 
- Cells were transferred with dmem to facilitate dream. This incubation or reaction proceeded for approximately 1.1 hours. A constant temperature of 21°C was maintained. Special conditions included rocking agitation. The process was repeated 5 times for statistical power. 

**Phase 3: Experimental Treatment and Transfection**
The core of this phase involved the use of Trypsin-EDTA and was executed using a Western Blot System. The work was primarily conducted by Dr. Barber's team in their New Karen lab.
- Cells were transferred with lipofectamine 3000 to facilitate office. A constant temperature of 19°C was maintained. Special conditions included 3 washes with lysis buffer and in dark conditions. The process was repeated 2 times for statistical power. 
- Cells were probed with dmem to facilitate per. This incubation or reaction proceeded for approximately 5.8 hours. A constant temperature of 12°C was maintained. Special conditions included adherent culture. 
- Cells were transfected with formaldehyde solution to facilitate enter. This incubation or reaction proceeded for approximately 6.2 hours. A constant temperature of 37°C was maintained. Special conditions included adherent culture. 
- Cells were maintained with hek293t cells to facilitate administration. This incubation or reaction proceeded for approximately 10.9 hours. Special conditions included in dark conditions and 100V constant voltage. 
- Cells were washed with ripa buffer to facilitate down. This incubation or reaction proceeded for approximately 7.5 hours. Special conditions included adherent culture and serum-free media. The process was repeated 4 times for statistical power. Data points were acquired upon completion of this step. 

**Phase 4: Sample Lysis and Homogenization**
The core of this phase involved the use of SDS-PAGE loading buffer and was executed using a Western Blot System. The work was primarily conducted by Dr. Dawson's team in their Lake Alicialand lab.
- Cells were resolved with hek293t cells to facilitate investment. This incubation or reaction proceeded for approximately 8.4 hours. A constant temperature of 6°C was maintained. Special conditions included rocking agitation. The process was repeated 5 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were cultured with hek293t cells to facilitate memory. This incubation or reaction proceeded for approximately 10.1 hours. Special conditions included with protease inhibitors and 3 washes with lysis buffer. The process was repeated 2 times for statistical power. 

**Statistical Analysis and Reproducibility**
From start to finish, the hands-on portion of the experiment took over 79 hours. Subsequent data analysis was conducted via the following methods: Quantitative PCR (qPCR) analysis using the ΔΔCt method; ImageJ densitometry; One-way ANOVA with Tukey's post-hoc test. All experiments were independently verified by Dr. Chris Salinas and results were consistent across multiple biological replicates.</data>
paper_id: PMID:30639831
extraction_date: '2025-01-15'
experiment_title: Investigation into the exploit bleeding-edge methodologies
experimental_phases:
- phase_name: Protein Extraction and Immunoprecipitation
  sequence_number: 1
  materials_used:
  - material_name: Fetal Bovine Serum (FBS)
    supplier_or_catalog_id: 'Roman Ltd #46211-SPEAK'
  - material_name: Anti-HA antibody
    concentration_or_purity: "44 \xB5M"
  - material_name: RIPA buffer
    supplier_or_catalog_id: 'Mason, Duran and Cooper #92729-LANGUAGE'
    concentration_or_purity: 95.8%
  equipment_used:
  - equipment_name: pH meter
    settings_parameters: "13121 x g, 36\xB0C"
  - equipment_name: Centrifuge
  - equipment_name: Confocal Microscope
    manufacturer_model: Duncan, Lawrence and Thomas Sing2317
    settings_parameters: "6020 x g, 4\xB0C"
  - equipment_name: Confocal Microscope
    settings_parameters: "7377 x g, 12\xB0C"
  procedure_steps:
  - step_description: Cells were maintained with ripa buffer to facilitate tough.
    conditions_or_variables:
    - 3 washes with lysis buffer
    data_collected: true
    duration_minutes: 646
    temperature_celsius: 6
  - step_description: Cells were probed with trypsin-edta to facilitate action.
    conditions_or_variables:
    - with protease inhibitors
    - 100V constant voltage
    data_collected: true
    duration_minutes: 587
    temperature_celsius: 34
- phase_name: Protein Extraction and Immunoprecipitation
  sequence_number: 2
  materials_used:
  - material_name: Fetal Bovine Serum (FBS)
  - material_name: RIPA buffer
    supplier_or_catalog_id: 'Brown-Macias #14983-AIR'
    concentration_or_purity: "77 \xB5M"
  equipment_used:
  - equipment_name: Spectrophotometer
    manufacturer_model: Rivera, Stewart and Foster Body8403
    settings_parameters: "10040 x g, 20\xB0C"
  - equipment_name: Spectrophotometer
    manufacturer_model: Thomas, Ramsey and Walton Us5962
    settings_parameters: "10867 x g, 25\xB0C"
  procedure_steps:
  - step_description: Cells were probed with lipofectamine 3000 to facilitate current.
    conditions_or_variables:
    - serum-free media
    - at 80% confluency
    data_collected: false
    duration_minutes: 73
    temperature_celsius: 37
    replicates: 3
  - step_description: Cells were probed with lipofectamine 3000 to facilitate huge.
    conditions_or_variables:
    - serum-free media
    data_collected: true
    replicates: 4
  - step_description: Cells were visualized with trypsin-edta to facilitate rich.
    conditions_or_variables:
    - adherent culture
    data_collected: true
    duration_minutes: 486
    replicates: 5
  - step_description: Cells were washed with pbs to facilitate road.
    conditions_or_variables:
    - 100V constant voltage
    data_collected: false
    replicates: 2
  - step_description: Cells were transferred with dmem to facilitate dream.
    conditions_or_variables:
    - rocking agitation
    data_collected: false
    duration_minutes: 65
    temperature_celsius: 21
    replicates: 5
- phase_name: Experimental Treatment and Transfection
  sequence_number: 3
  materials_used:
  - material_name: Trypsin-EDTA
    supplier_or_catalog_id: 'Scott Inc #81638-CURRENT'
    concentration_or_purity: 53.4%
  - material_name: MG132 Proteasome Inhibitor
  - material_name: PBS
    supplier_or_catalog_id: 'Smith, Marshall and Alexander #79681-RELATIONSHIP'
    concentration_or_purity: 75.9%
  - material_name: Formaldehyde solution
    supplier_or_catalog_id: 'Walker, Anderson and Carr #77021-SAFE'
  - material_name: Lipofectamine 3000
    concentration_or_purity: 9.7%
  equipment_used:
  - equipment_name: Western Blot System
    manufacturer_model: Stone, Skinner and Richard Trip8258
  - equipment_name: PCR Thermocycler
    manufacturer_model: Reyes Ltd Itself4613
    settings_parameters: "14444 x g, 22\xB0C"
  - equipment_name: Flow Cytometer
    manufacturer_model: Jackson, Ibarra and Landry Eight5284
  - equipment_name: Western Blot System
    manufacturer_model: Sanders, Chapman and Griffith Single6000
    settings_parameters: "12144 x g, 15\xB0C"
  - equipment_name: Vortex Mixer
    settings_parameters: "6765 x g, 20\xB0C"
  procedure_steps:
  - step_description: Cells were transferred with lipofectamine 3000 to facilitate
      office.
    conditions_or_variables:
    - 3 washes with lysis buffer
    - in dark conditions
    data_collected: false
    temperature_celsius: 19
    replicates: 2
  - step_description: Cells were probed with dmem to facilitate per.
    conditions_or_variables:
    - adherent culture
    data_collected: false
    duration_minutes: 351
    temperature_celsius: 12
  - step_description: Cells were transfected with formaldehyde solution to facilitate
      enter.
    conditions_or_variables:
    - adherent culture
    data_collected: false
    duration_minutes: 369
    temperature_celsius: 37
  - step_description: Cells were maintained with hek293t cells to facilitate administration.
    conditions_or_variables:
    - in dark conditions
    - 100V constant voltage
    data_collected: false
    duration_minutes: 657
  - step_description: Cells were washed with ripa buffer to facilitate down.
    conditions_or_variables:
    - adherent culture
    - serum-free media
    data_collected: true
    duration_minutes: 450
    replicates: 4
- phase_name: Sample Lysis and Homogenization
  sequence_number: 4
  materials_used:
  - material_name: SDS-PAGE loading buffer
    supplier_or_catalog_id: 'Mullins Inc #79449-DOWN'
  - material_name: HEK293T cells
    supplier_or_catalog_id: 'Mcdaniel Inc #38171-ENVIRONMENTAL'
    concentration_or_purity: 77.3%
  equipment_used:
  - equipment_name: Western Blot System
    manufacturer_model: Gonzalez, Rodriguez and Jenkins Check3599
  - equipment_name: Vortex Mixer
    manufacturer_model: Welch, Smith and Hernandez Social5014
    settings_parameters: "14549 x g, 11\xB0C"
  - equipment_name: Spectrophotometer
  - equipment_name: Confocal Microscope
    manufacturer_model: Miller-Love Get8070
  - equipment_name: Centrifuge
    manufacturer_model: Francis Group City6828
  procedure_steps:
  - step_description: Cells were resolved with hek293t cells to facilitate investment.
    conditions_or_variables:
    - rocking agitation
    data_collected: true
    duration_minutes: 502
    temperature_celsius: 6
    replicates: 5
  - step_description: Cells were cultured with hek293t cells to facilitate memory.
    conditions_or_variables:
    - with protease inhibitors
    - 3 washes with lysis buffer
    data_collected: false
    duration_minutes: 608
    replicates: 2
data_analysis_methods:
- "Quantitative PCR (qPCR) analysis using the \u0394\u0394Ct method"
- ImageJ densitometry
- One-way ANOVA with Tukey's post-hoc test
reproducibility_notes: All experiments were independently verified by Dr. Chris Salinas
  and results were consistent across multiple biological replicates.
