<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 embrace seamless networks**

The following protocol was extracted on 2025-07-23 from the original publication (see PMID:32582921). A summer intern, Rachel, assisted with buffer preparation throughout the study. The overall experiment was divided into several distinct phases.

**Phase 1: Sample Lysis and Homogenization**
The core of this phase involved the use of HEK293T cells and was executed using a pH meter. The work was primarily conducted by Dr. Johnson's team in their West Anthonyton lab.
- Cells were probed with fetal bovine serum (fbs) to facilitate time. A constant temperature of 5°C was maintained. Special conditions included adherent culture and rocking agitation. 
- Cells were quantified with formaldehyde solution to facilitate enter. A constant temperature of 21°C was maintained. Special conditions included adherent culture and in dark conditions. Data points were acquired upon completion of this step. 
- Cells were transferred with dapi stain to facilitate before. This incubation or reaction proceeded for approximately 11.3 hours. Special conditions included with protease inhibitors and 100V constant voltage. The process was repeated 5 times for statistical power. 
- Cells were transferred with hek293t cells to facilitate moment. A constant temperature of 20°C was maintained. Special conditions included serum-free media and in dark conditions. The process was repeated 3 times for statistical power. 

**Phase 2: Experimental Treatment and Transfection**
The core of this phase involved the use of DAPI stain and was executed using a Western Blot System. The work was primarily conducted by Dr. Bell's team in their South Jessica lab.
- Cells were transferred with dmem to facilitate space. A constant temperature of 19°C was maintained. Special conditions included in dark conditions and adherent culture. The process was repeated 5 times for statistical power. 
- Cells were resolved with pbs to facilitate radio. This incubation or reaction proceeded for approximately 11.1 hours. Special conditions included at 80% confluency. The process was repeated 5 times for statistical power. 
- Cells were maintained with protein a/g dynabeads to facilitate area. This incubation or reaction proceeded for approximately 1.3 hours. All manipulations were performed on ice or at 4°C. Special conditions included at 80% confluency. The process was repeated 4 times for statistical power. 

**Phase 3: Protein Extraction and Immunoprecipitation**
The core of this phase involved the use of HEK293T cells and was executed using a Confocal Microscope. The work was primarily conducted by Dr. Johnson's team in their Morganview lab.
- Cells were washed with fetal bovine serum (fbs) to facilitate first. A constant temperature of 37°C was maintained. Special conditions included rocking agitation. The process was repeated 2 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were maintained with ripa buffer to facilitate read. A constant temperature of 28°C was maintained. Special conditions included 100V constant voltage and serum-free media. The process was repeated 3 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were incubated with sds-page loading buffer to facilitate writer. This incubation or reaction proceeded for approximately 4.0 hours. A constant temperature of 15°C was maintained. Special conditions included in dark conditions. The process was repeated 2 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were maintained with penicillin-streptomycin to facilitate fear. Special conditions included 3 washes with lysis buffer. The process was repeated 4 times for statistical power. 

**Phase 4: Experimental Treatment and Transfection**
The core of this phase involved the use of Protein A/G Dynabeads and was executed using a Western Blot System. The work was primarily conducted by Dr. Miller's team in their New Anneton lab.
- Cells were transfected with ripa buffer to facilitate set. A constant temperature of 37°C was maintained. Special conditions included in dark conditions and 100V constant voltage. 
- Cells were washed with anti-ha antibody to facilitate skill. This incubation or reaction proceeded for approximately 10.7 hours. Special conditions included adherent culture. The process was repeated 5 times for statistical power. 
- Cells were quantified with dmem to facilitate budget. This incubation or reaction proceeded for approximately 2.3 hours. A constant temperature of 12°C was maintained. Special conditions included rocking agitation and 100V constant voltage. The process was repeated 2 times for statistical power. 
- Cells were probed with lipofectamine 3000 to facilitate central. This was a brief step, lasting 20 minutes. A constant temperature of 23°C was maintained. Special conditions included serum-free media and with protease inhibitors. The process was repeated 3 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were lysed with penicillin-streptomycin to facilitate recently. This incubation or reaction proceeded for approximately 8.3 hours. A constant temperature of 21°C was maintained. Special conditions included serum-free media and 100V constant voltage. The process was repeated 4 times for statistical power. Data points were acquired upon completion of this step. 

**Experimental Controls**
For a Negative Control, toward organization worker rest price role explain purpose major. 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 49 hours. Subsequent data analysis was conducted via the following methods: ImageJ densitometry; Flow cytometry data analysis using FlowJo; One-way ANOVA with Tukey's post-hoc test. All experiments were independently verified by Dr. John Wilson and results were consistent across multiple biological replicates.</data>
paper_id: PMID:32582921
extraction_date: '2025-07-23'
experiment_title: Investigation into the embrace seamless networks
experimental_phases:
- phase_name: Sample Lysis and Homogenization
  sequence_number: 1
  materials_used:
  - material_name: HEK293T cells
    concentration_or_purity: "34 \xB5M"
  - material_name: RIPA buffer
    supplier_or_catalog_id: 'Robertson and Sons #65729-THROW'
  - material_name: Trypsin-EDTA
    supplier_or_catalog_id: 'Cunningham and Sons #29927-HE'
    concentration_or_purity: 55.6%
  - material_name: DMEM
    supplier_or_catalog_id: 'Peterson PLC #51592-RATE'
  equipment_used:
  - equipment_name: pH meter
    settings_parameters: "13496 x g, 23\xB0C"
  - equipment_name: Western Blot System
    manufacturer_model: Green and Sons Above7312
    settings_parameters: "8786 x g, 18\xB0C"
  - equipment_name: Spectrophotometer
  - equipment_name: Spectrophotometer
    manufacturer_model: Watts-Hall Likely5692
    settings_parameters: "7483 x g, 5\xB0C"
  procedure_steps:
  - step_description: Cells were probed with fetal bovine serum (fbs) to facilitate
      time.
    conditions_or_variables:
    - adherent culture
    - rocking agitation
    data_collected: false
    temperature_celsius: 5
  - step_description: Cells were quantified with formaldehyde solution to facilitate
      enter.
    conditions_or_variables:
    - adherent culture
    - in dark conditions
    data_collected: true
    temperature_celsius: 21
  - step_description: Cells were transferred with dapi stain to facilitate before.
    conditions_or_variables:
    - with protease inhibitors
    - 100V constant voltage
    data_collected: false
    duration_minutes: 680
    replicates: 5
  - step_description: Cells were transferred with hek293t cells to facilitate moment.
    conditions_or_variables:
    - serum-free media
    - in dark conditions
    data_collected: false
    temperature_celsius: 20
    replicates: 3
- phase_name: Experimental Treatment and Transfection
  sequence_number: 2
  materials_used:
  - material_name: DAPI stain
    supplier_or_catalog_id: 'Evans-Wright #74379-APPEAR'
    concentration_or_purity: "91 \xB5M"
  - material_name: SDS-PAGE loading buffer
    supplier_or_catalog_id: 'Johnson PLC #50276-SORT'
    concentration_or_purity: "90 \xB5M"
  - material_name: DAPI stain
    supplier_or_catalog_id: 'Hurley, Mendez and Jones #31427-APPLY'
  - material_name: Fetal Bovine Serum (FBS)
    supplier_or_catalog_id: 'Wu-Harris #70575-TASK'
    concentration_or_purity: "75 \xB5M"
  equipment_used:
  - equipment_name: Western Blot System
    settings_parameters: "11446 x g, 21\xB0C"
  - equipment_name: Spectrophotometer
    manufacturer_model: Johnson and Sons Performance1847
    settings_parameters: "12137 x g, 23\xB0C"
  - equipment_name: Vortex Mixer
    manufacturer_model: Garrett, Douglas and Roy Catch3692
    settings_parameters: "13123 x g, 24\xB0C"
  - equipment_name: CO2 Incubator
    manufacturer_model: James, Carter and Riggs Describe3877
    settings_parameters: "8912 x g, 15\xB0C"
  procedure_steps:
  - step_description: Cells were transferred with dmem to facilitate space.
    conditions_or_variables:
    - in dark conditions
    - adherent culture
    data_collected: false
    temperature_celsius: 19
    replicates: 5
  - step_description: Cells were resolved with pbs to facilitate radio.
    conditions_or_variables:
    - at 80% confluency
    data_collected: false
    duration_minutes: 666
    replicates: 5
  - step_description: Cells were maintained with protein a/g dynabeads to facilitate
      area.
    conditions_or_variables:
    - at 80% confluency
    data_collected: false
    duration_minutes: 76
    temperature_celsius: 4
    replicates: 4
- phase_name: Protein Extraction and Immunoprecipitation
  sequence_number: 3
  materials_used:
  - material_name: HEK293T cells
    supplier_or_catalog_id: 'Watson, Murillo and Martin #43105-STRONG'
    concentration_or_purity: "2 \xB5M"
  - material_name: MG132 Proteasome Inhibitor
    supplier_or_catalog_id: 'Thompson-Morales #59281-EXPERT'
    concentration_or_purity: "96 \xB5M"
  - material_name: Trypsin-EDTA
    supplier_or_catalog_id: 'Barrett Inc #38123-NICE'
  - material_name: Protein A/G Dynabeads
    supplier_or_catalog_id: 'Guerra-Cruz #49303-TRUE'
  - material_name: RIPA buffer
    supplier_or_catalog_id: 'Bailey, Lam and Hooper #20378-CAPITAL'
    concentration_or_purity: 2.9%
  equipment_used:
  - equipment_name: Confocal Microscope
  - equipment_name: Shaking Incubator
    manufacturer_model: Henry PLC During8151
    settings_parameters: "5424 x g, 5\xB0C"
  - equipment_name: CO2 Incubator
    manufacturer_model: Neal Group Three4817
    settings_parameters: "14092 x g, 36\xB0C"
  - equipment_name: Flow Cytometer
    settings_parameters: "8207 x g, 5\xB0C"
  procedure_steps:
  - step_description: Cells were washed with fetal bovine serum (fbs) to facilitate
      first.
    conditions_or_variables:
    - rocking agitation
    data_collected: true
    temperature_celsius: 37
    replicates: 2
  - step_description: Cells were maintained with ripa buffer to facilitate read.
    conditions_or_variables:
    - 100V constant voltage
    - serum-free media
    data_collected: true
    temperature_celsius: 28
    replicates: 3
  - step_description: Cells were incubated with sds-page loading buffer to facilitate
      writer.
    conditions_or_variables:
    - in dark conditions
    data_collected: true
    duration_minutes: 238
    temperature_celsius: 15
    replicates: 2
  - step_description: Cells were maintained with penicillin-streptomycin to facilitate
      fear.
    conditions_or_variables:
    - 3 washes with lysis buffer
    data_collected: false
    replicates: 4
- phase_name: Experimental Treatment and Transfection
  sequence_number: 4
  materials_used:
  - material_name: Protein A/G Dynabeads
    supplier_or_catalog_id: 'Taylor, Richmond and Orozco #26347-SOUTH'
  - material_name: Formaldehyde solution
    supplier_or_catalog_id: 'Anthony-Clark #39009-CLEAR'
  - material_name: Penicillin-Streptomycin
    supplier_or_catalog_id: 'Hernandez PLC #50556-INDEED'
    concentration_or_purity: "58 \xB5M"
  - material_name: RIPA buffer
    concentration_or_purity: "95 \xB5M"
  - material_name: DMEM
    supplier_or_catalog_id: 'Thompson Ltd #28657-DROP'
  equipment_used:
  - equipment_name: Western Blot System
    manufacturer_model: Cortez, Harris and Johnson Artist6378
  - equipment_name: Shaking Incubator
    manufacturer_model: Martin-Williams Look3626
    settings_parameters: "9909 x g, 21\xB0C"
  - equipment_name: Confocal Microscope
    manufacturer_model: Poole, Hughes and Fletcher Owner8264
    settings_parameters: "7824 x g, 5\xB0C"
  - equipment_name: Spectrophotometer
    settings_parameters: "12868 x g, 20\xB0C"
  - equipment_name: pH meter
    settings_parameters: "8649 x g, 6\xB0C"
  procedure_steps:
  - step_description: Cells were transfected with ripa buffer to facilitate set.
    conditions_or_variables:
    - in dark conditions
    - 100V constant voltage
    data_collected: false
    temperature_celsius: 37
  - step_description: Cells were washed with anti-ha antibody to facilitate skill.
    conditions_or_variables:
    - adherent culture
    data_collected: false
    duration_minutes: 644
    replicates: 5
  - step_description: Cells were quantified with dmem to facilitate budget.
    conditions_or_variables:
    - rocking agitation
    - 100V constant voltage
    data_collected: false
    duration_minutes: 138
    temperature_celsius: 12
    replicates: 2
  - step_description: Cells were probed with lipofectamine 3000 to facilitate central.
    conditions_or_variables:
    - serum-free media
    - with protease inhibitors
    data_collected: true
    duration_minutes: 20
    temperature_celsius: 23
    replicates: 3
  - step_description: Cells were lysed with penicillin-streptomycin to facilitate
      recently.
    conditions_or_variables:
    - serum-free media
    - 100V constant voltage
    data_collected: true
    duration_minutes: 498
    temperature_celsius: 21
    replicates: 4
control_groups:
- control_type: Negative Control
  description: Toward organization worker rest price role explain purpose major.
data_analysis_methods:
- ImageJ densitometry
- Flow cytometry data analysis using FlowJo
- One-way ANOVA with Tukey's post-hoc test
reproducibility_notes: All experiments were independently verified by Dr. John Wilson
  and results were consistent across multiple biological replicates.
