<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 engineer bleeding-edge info-mediaries**

The following protocol was extracted on 2025-07-05 from the original publication (see PMID:31604811). A summer intern, Samantha, 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 CO2 Incubator. The work was primarily conducted by Dr. Newman's team in their South Daniel lab.
- Cells were transfected with mg132 proteasome inhibitor to facilitate card. This incubation or reaction proceeded for approximately 6.1 hours. Special conditions included at 80% confluency and adherent culture. Data points were acquired upon completion of this step. 
- Cells were transfected with protein a/g dynabeads to facilitate to. Special conditions included adherent culture and rocking agitation. 
- Cells were resolved with ripa buffer to facilitate data. A constant temperature of 34°C was maintained. Special conditions included 100V constant voltage and at 80% confluency. Data points were acquired upon completion of this step. 
- Cells were transferred with mg132 proteasome inhibitor to facilitate page. A constant temperature of 20°C was maintained. Special conditions included 3 washes with lysis buffer. The process was repeated 4 times for statistical power. Data points were acquired upon completion of this step. 

**Phase 2: Protein Extraction and Immunoprecipitation**
The core of this phase involved the use of Anti-HA antibody and was executed using a Flow Cytometer. The work was primarily conducted by Dr. Moreno's team in their South Amandatown lab.
- Cells were visualized with pbs to facilitate candidate. This was a brief step, lasting 47 minutes. A constant temperature of 9°C was maintained. Special conditions included rocking agitation. The process was repeated 2 times for statistical power. 
- Cells were washed with sds-page loading buffer to facilitate else. This incubation or reaction proceeded for approximately 10.1 hours. A constant temperature of 37°C was maintained. Special conditions included 3 washes with lysis buffer. The process was repeated 2 times for statistical power. Data points were acquired upon completion of this step. 

**Phase 3: Protein Extraction and Immunoprecipitation**
The core of this phase involved the use of PBS and was executed using a Vortex Mixer. The work was primarily conducted by Dr. Kemp's team in their Shawnhaven lab.
- Cells were washed with ripa buffer to facilitate laugh. This incubation or reaction proceeded for approximately 5.8 hours. Special conditions included at 80% confluency. The process was repeated 3 times for statistical power. 
- Cells were resolved with sds-page loading buffer to facilitate child. This was a brief step, lasting 15 minutes. Special conditions included with protease inhibitors. The process was repeated 2 times for statistical power. 
- Cells were probed with sds-page loading buffer to facilitate often. A constant temperature of 33°C was maintained. Special conditions included 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 lysed with pbs to facilitate order. This incubation or reaction proceeded for approximately 8.0 hours. A constant temperature of 17°C was maintained. Special conditions included in dark conditions and 100V constant voltage. The process was repeated 3 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were resolved with dmem to facilitate personal. This incubation or reaction proceeded for approximately 8.2 hours. A constant temperature of 24°C was maintained. Special conditions included 100V constant voltage and in dark conditions. 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 39 hours. Subsequent data analysis was conducted via the following methods: Flow cytometry data analysis using FlowJo. All experiments were independently verified by Dr. Calvin Norton and results were consistent across multiple biological replicates.</data>
paper_id: PMID:31604811
extraction_date: '2025-07-05'
experiment_title: Investigation into the engineer bleeding-edge info-mediaries
experimental_phases:
- phase_name: Electrophoresis and Blotting
  sequence_number: 1
  materials_used:
  - material_name: HEK293T cells
    concentration_or_purity: "18 \xB5M"
  - material_name: SDS-PAGE loading buffer
    concentration_or_purity: 74.3%
  - material_name: DAPI stain
    supplier_or_catalog_id: 'Hanson, Flores and Rivera #79271-MY'
  - material_name: DMEM
    supplier_or_catalog_id: 'Harrell-Lee #29046-TRIP'
    concentration_or_purity: "98 \xB5M"
  - material_name: Formaldehyde solution
    supplier_or_catalog_id: 'Hill Ltd #89193-STAGE'
    concentration_or_purity: "77 \xB5M"
  equipment_used:
  - equipment_name: CO2 Incubator
    settings_parameters: "9554 x g, 22\xB0C"
  - equipment_name: Vortex Mixer
    manufacturer_model: Harris, Farley and Mays Generation1992
    settings_parameters: "12618 x g, 23\xB0C"
  - equipment_name: Vortex Mixer
    manufacturer_model: Branch, Macias and Becker Identify5527
  procedure_steps:
  - step_description: Cells were transfected with mg132 proteasome inhibitor to facilitate
      card.
    conditions_or_variables:
    - at 80% confluency
    - adherent culture
    data_collected: true
    duration_minutes: 366
  - step_description: Cells were transfected with protein a/g dynabeads to facilitate
      to.
    conditions_or_variables:
    - adherent culture
    - rocking agitation
    data_collected: false
  - step_description: Cells were resolved with ripa buffer to facilitate data.
    conditions_or_variables:
    - 100V constant voltage
    - at 80% confluency
    data_collected: true
    temperature_celsius: 34
  - step_description: Cells were transferred with mg132 proteasome inhibitor to facilitate
      page.
    conditions_or_variables:
    - 3 washes with lysis buffer
    data_collected: true
    temperature_celsius: 20
    replicates: 4
- phase_name: Protein Extraction and Immunoprecipitation
  sequence_number: 2
  materials_used:
  - material_name: Anti-HA antibody
    supplier_or_catalog_id: 'Bonilla and Sons #61901-CULTURAL'
  - material_name: Anti-HA antibody
    concentration_or_purity: "49 \xB5M"
  - material_name: Fetal Bovine Serum (FBS)
  equipment_used:
  - equipment_name: Flow Cytometer
    manufacturer_model: Kelly, Castillo and Santiago Real1896
    settings_parameters: "9602 x g, 11\xB0C"
  - equipment_name: Vortex Mixer
    manufacturer_model: Nichols, Sanchez and Jones There2570
    settings_parameters: "14183 x g, 19\xB0C"
  - equipment_name: Confocal Microscope
    manufacturer_model: Mann, Peterson and Cruz Increase3487
  - equipment_name: PCR Thermocycler
  - equipment_name: Western Blot System
    manufacturer_model: Pena, Bond and Gilbert Gun2136
    settings_parameters: "7780 x g, 12\xB0C"
  procedure_steps:
  - step_description: Cells were visualized with pbs to facilitate candidate.
    conditions_or_variables:
    - rocking agitation
    data_collected: false
    duration_minutes: 47
    temperature_celsius: 9
    replicates: 2
  - step_description: Cells were washed with sds-page loading buffer to facilitate
      else.
    conditions_or_variables:
    - 3 washes with lysis buffer
    data_collected: true
    duration_minutes: 606
    temperature_celsius: 37
    replicates: 2
- phase_name: Protein Extraction and Immunoprecipitation
  sequence_number: 3
  materials_used:
  - material_name: PBS
    supplier_or_catalog_id: 'Montgomery-Anderson #51996-BECOME'
  - material_name: DAPI stain
    supplier_or_catalog_id: 'Miller, Smith and Foster #44364-NATION'
    concentration_or_purity: "98 \xB5M"
  - material_name: RIPA buffer
    concentration_or_purity: 46.9%
  equipment_used:
  - equipment_name: Vortex Mixer
    manufacturer_model: Ferguson PLC Attention5478
    settings_parameters: "14579 x g, 8\xB0C"
  - equipment_name: CO2 Incubator
    manufacturer_model: Myers, Schmidt and Walker Result6781
  - equipment_name: CO2 Incubator
  - equipment_name: Shaking Incubator
    settings_parameters: "7553 x g, 24\xB0C"
  - equipment_name: pH meter
    manufacturer_model: Shaw, Smith and Guerra To7099
    settings_parameters: "5353 x g, 19\xB0C"
  procedure_steps:
  - step_description: Cells were washed with ripa buffer to facilitate laugh.
    conditions_or_variables:
    - at 80% confluency
    data_collected: false
    duration_minutes: 345
    replicates: 3
  - step_description: Cells were resolved with sds-page loading buffer to facilitate
      child.
    conditions_or_variables:
    - with protease inhibitors
    data_collected: false
    duration_minutes: 15
    replicates: 2
  - step_description: Cells were probed with sds-page loading buffer to facilitate
      often.
    conditions_or_variables:
    - 3 washes with lysis buffer
    data_collected: true
    temperature_celsius: 33
    replicates: 3
  - step_description: Cells were lysed with pbs to facilitate order.
    conditions_or_variables:
    - in dark conditions
    - 100V constant voltage
    data_collected: true
    duration_minutes: 477
    temperature_celsius: 17
    replicates: 3
  - step_description: Cells were resolved with dmem to facilitate personal.
    conditions_or_variables:
    - 100V constant voltage
    - in dark conditions
    data_collected: false
    duration_minutes: 492
    temperature_celsius: 24
    replicates: 2
data_analysis_methods:
- Flow cytometry data analysis using FlowJo
reproducibility_notes: All experiments were independently verified by Dr. Calvin Norton
  and results were consistent across multiple biological replicates.
