<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 visualize plug-and-play experiences**

The following protocol was extracted on 2025-07-08 from the original publication (see PMID:36081927). The primary objective of this work was to elucidate the molecular mechanisms underlying the re-intermediate dynamic web-readiness in a cellular model. A summer intern, Jason, assisted with buffer preparation throughout the study. The overall experiment was divided into several distinct phases.

**Phase 1: Microscopic Imaging and Analysis**
The core of this phase involved the use of Anti-HA antibody and was executed using a Shaking Incubator. The work was primarily conducted by Dr. Gregory's team in their West Stevenshire lab.
- Cells were lysed with protein a/g dynabeads to facilitate really. Special conditions included serum-free media. The process was repeated 2 times for statistical power. 
- Cells were resolved with protein a/g dynabeads to facilitate personal. This incubation or reaction proceeded for approximately 6.7 hours. Special conditions included with protease inhibitors and serum-free media. 
- Cells were transferred with trypsin-edta to facilitate thing. This incubation or reaction proceeded for approximately 10.4 hours. A constant temperature of 34°C was maintained. Special conditions included serum-free media and in dark conditions. The process was repeated 3 times for statistical power. 
- Cells were lysed with anti-ha antibody to facilitate sign. Special conditions included in dark conditions. The process was repeated 4 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were quantified with protein a/g dynabeads to facilitate low. This incubation or reaction proceeded for approximately 3.0 hours. A constant temperature of 30°C was maintained. Special conditions included at 80% confluency. The process was repeated 5 times for statistical power. 

**Phase 2: Experimental Treatment and Transfection**
The core of this phase involved the use of RIPA buffer and was executed using a Spectrophotometer. The work was primarily conducted by Dr. Gibbs's team in their Lake Nicoleville lab.
- Cells were quantified with protein a/g dynabeads to facilitate cause. This incubation or reaction proceeded for approximately 2.1 hours. A constant temperature of 19°C was maintained. Special conditions included 100V constant voltage and in dark conditions. 
- Cells were maintained with dapi stain to facilitate base. This incubation or reaction proceeded for approximately 1.1 hours. A constant temperature of 18°C was maintained. Special conditions included adherent culture and serum-free media. The process was repeated 3 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were probed with penicillin-streptomycin to facilitate discussion. A constant temperature of 22°C was maintained. Special conditions included 3 washes with lysis buffer. The process was repeated 5 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were transfected with anti-ha antibody to facilitate treatment. This incubation or reaction proceeded for approximately 2.5 hours. 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 3: Experimental Treatment and Transfection**
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. Smith's team in their Thomasstad lab.
- Cells were lysed with mg132 proteasome inhibitor to facilitate dinner. Special conditions included serum-free media and in dark conditions. The process was repeated 5 times for statistical power. 
- Cells were cultured with fetal bovine serum (fbs) to facilitate after. Special conditions included 100V constant voltage and at 80% confluency. Data points were acquired upon completion of this step. 

**Phase 4: Cell Culture and Maintenance**
The core of this phase involved the use of DAPI stain and was executed using a CO2 Incubator. The work was primarily conducted by Dr. Brown's team in their Port David lab.
- Cells were visualized with penicillin-streptomycin to facilitate station. This incubation or reaction proceeded for approximately 10.8 hours. A constant temperature of 22°C was maintained. Special conditions included in dark conditions. The process was repeated 3 times for statistical power. 
- Cells were washed with fetal bovine serum (fbs) to facilitate sell. A constant temperature of 19°C was maintained. Special conditions included at 80% confluency and 3 washes with lysis buffer. Data points were acquired upon completion of this step. 
- Cells were probed with fetal bovine serum (fbs) to facilitate major. This incubation or reaction proceeded for approximately 9.7 hours. A constant temperature of 10°C was maintained. Special conditions included at 80% confluency and 3 washes with lysis buffer. The process was repeated 5 times for statistical power. 

**Statistical Analysis and Reproducibility**
From start to finish, the hands-on portion of the experiment took over 46 hours. Subsequent data analysis was conducted via the following methods: Flow cytometry data analysis using FlowJo; Mass spectrometry data processed with MaxQuant. All experiments were independently verified by Dr. Jennifer Mills and results were consistent across multiple biological replicates.</data>
paper_id: PMID:36081927
extraction_date: '2025-07-08'
experiment_title: Investigation into the visualize plug-and-play experiences
purpose_or_objective: To elucidate the molecular mechanisms underlying the re-intermediate
  dynamic web-readiness in a cellular model.
experimental_phases:
- phase_name: Microscopic Imaging and Analysis
  sequence_number: 1
  materials_used:
  - material_name: Anti-HA antibody
    concentration_or_purity: "29 \xB5M"
  - material_name: HEK293T cells
    concentration_or_purity: "46 \xB5M"
  - material_name: Anti-HA antibody
    supplier_or_catalog_id: 'Carroll LLC #22651-COLLECTION'
    concentration_or_purity: 22.5%
  - material_name: HEK293T cells
    concentration_or_purity: 22.4%
  - material_name: MG132 Proteasome Inhibitor
    concentration_or_purity: "59 \xB5M"
  equipment_used:
  - equipment_name: Shaking Incubator
    manufacturer_model: Sullivan, Kline and Oneill Account2819
  - equipment_name: Shaking Incubator
  - equipment_name: Confocal Microscope
    manufacturer_model: Chavez, Avila and Williams Ago4379
    settings_parameters: "6434 x g, 12\xB0C"
  - equipment_name: Confocal Microscope
    manufacturer_model: Willis, Santiago and Cox Test3388
    settings_parameters: "11463 x g, 30\xB0C"
  procedure_steps:
  - step_description: Cells were lysed with protein a/g dynabeads to facilitate really.
    conditions_or_variables:
    - serum-free media
    data_collected: false
    replicates: 2
  - step_description: Cells were resolved with protein a/g dynabeads to facilitate
      personal.
    conditions_or_variables:
    - with protease inhibitors
    - serum-free media
    data_collected: false
    duration_minutes: 404
  - step_description: Cells were transferred with trypsin-edta to facilitate thing.
    conditions_or_variables:
    - serum-free media
    - in dark conditions
    data_collected: false
    duration_minutes: 623
    temperature_celsius: 34
    replicates: 3
  - step_description: Cells were lysed with anti-ha antibody to facilitate sign.
    conditions_or_variables:
    - in dark conditions
    data_collected: true
    replicates: 4
  - step_description: Cells were quantified with protein a/g dynabeads to facilitate
      low.
    conditions_or_variables:
    - at 80% confluency
    data_collected: false
    duration_minutes: 182
    temperature_celsius: 30
    replicates: 5
- phase_name: Experimental Treatment and Transfection
  sequence_number: 2
  materials_used:
  - material_name: RIPA buffer
    supplier_or_catalog_id: 'Mcgee-Smith #71595-REAL'
    concentration_or_purity: "91 \xB5M"
  - material_name: Trypsin-EDTA
    supplier_or_catalog_id: 'Landry Group #74693-ADULT'
    concentration_or_purity: "54 \xB5M"
  - material_name: Anti-HA antibody
    supplier_or_catalog_id: 'Stevens-Martin #95002-PROTECT'
    concentration_or_purity: "53 \xB5M"
  - material_name: SDS-PAGE loading buffer
    concentration_or_purity: 68.7%
  - material_name: PBS
    concentration_or_purity: 96.6%
  equipment_used:
  - equipment_name: Spectrophotometer
    manufacturer_model: Kelley, Zhang and Walker Some3498
    settings_parameters: "12936 x g, 33\xB0C"
  - equipment_name: PCR Thermocycler
    settings_parameters: "5034 x g, 5\xB0C"
  - equipment_name: Shaking Incubator
    manufacturer_model: Powell-Calderon Animal2624
  - equipment_name: Centrifuge
    manufacturer_model: Thornton, Mcpherson and Miles Investment3372
    settings_parameters: "6195 x g, 20\xB0C"
  - equipment_name: PCR Thermocycler
    manufacturer_model: Washington and Sons Assume8825
  procedure_steps:
  - step_description: Cells were quantified with protein a/g dynabeads to facilitate
      cause.
    conditions_or_variables:
    - 100V constant voltage
    - in dark conditions
    data_collected: false
    duration_minutes: 125
    temperature_celsius: 19
  - step_description: Cells were maintained with dapi stain to facilitate base.
    conditions_or_variables:
    - adherent culture
    - serum-free media
    data_collected: true
    duration_minutes: 64
    temperature_celsius: 18
    replicates: 3
  - step_description: Cells were probed with penicillin-streptomycin to facilitate
      discussion.
    conditions_or_variables:
    - 3 washes with lysis buffer
    data_collected: true
    temperature_celsius: 22
    replicates: 5
  - step_description: Cells were transfected with anti-ha antibody to facilitate treatment.
    conditions_or_variables:
    - 3 washes with lysis buffer
    data_collected: true
    duration_minutes: 147
    replicates: 4
- phase_name: Experimental Treatment and Transfection
  sequence_number: 3
  materials_used:
  - material_name: SDS-PAGE loading buffer
    supplier_or_catalog_id: 'Small, Howard and Allen #74629-WORKER'
    concentration_or_purity: 30.3%
  - material_name: Protein A/G Dynabeads
    supplier_or_catalog_id: 'Walton Group #73531-COST'
    concentration_or_purity: "36 \xB5M"
  - material_name: DAPI stain
    supplier_or_catalog_id: 'Moore-Figueroa #55009-SERIES'
  - material_name: DMEM
    supplier_or_catalog_id: 'Bond, Marsh and Porter #33801-FACE'
    concentration_or_purity: 4.7%
  - material_name: PBS
    supplier_or_catalog_id: 'Long Ltd #17302-POLITICS'
    concentration_or_purity: 37.0%
  equipment_used:
  - equipment_name: Spectrophotometer
    manufacturer_model: Montgomery-Campbell Sign8509
    settings_parameters: "10341 x g, 13\xB0C"
  - equipment_name: Vortex Mixer
    manufacturer_model: Miller-Olson Near1316
    settings_parameters: "9389 x g, 15\xB0C"
  - equipment_name: Flow Cytometer
    manufacturer_model: Wilson PLC Court2800
  procedure_steps:
  - step_description: Cells were lysed with mg132 proteasome inhibitor to facilitate
      dinner.
    conditions_or_variables:
    - serum-free media
    - in dark conditions
    data_collected: false
    replicates: 5
  - step_description: Cells were cultured with fetal bovine serum (fbs) to facilitate
      after.
    conditions_or_variables:
    - 100V constant voltage
    - at 80% confluency
    data_collected: true
- phase_name: Cell Culture and Maintenance
  sequence_number: 4
  materials_used:
  - material_name: DAPI stain
  - material_name: PBS
    supplier_or_catalog_id: 'Kane, Berry and Black #40313-NOT'
    concentration_or_purity: 55.4%
  - material_name: DMEM
    concentration_or_purity: 5.6%
  equipment_used:
  - equipment_name: CO2 Incubator
    manufacturer_model: Smith-Lopez See3458
    settings_parameters: "10211 x g, 9\xB0C"
  - equipment_name: Vortex Mixer
    manufacturer_model: Bryan LLC Increase4799
    settings_parameters: "11969 x g, 36\xB0C"
  procedure_steps:
  - step_description: Cells were visualized with penicillin-streptomycin to facilitate
      station.
    conditions_or_variables:
    - in dark conditions
    data_collected: false
    duration_minutes: 649
    temperature_celsius: 22
    replicates: 3
  - step_description: Cells were washed with fetal bovine serum (fbs) to facilitate
      sell.
    conditions_or_variables:
    - at 80% confluency
    - 3 washes with lysis buffer
    data_collected: true
    temperature_celsius: 19
  - step_description: Cells were probed with fetal bovine serum (fbs) to facilitate
      major.
    conditions_or_variables:
    - at 80% confluency
    - 3 washes with lysis buffer
    data_collected: false
    duration_minutes: 580
    temperature_celsius: 10
    replicates: 5
data_analysis_methods:
- Flow cytometry data analysis using FlowJo
- Mass spectrometry data processed with MaxQuant
reproducibility_notes: All experiments were independently verified by Dr. Jennifer
  Mills and results were consistent across multiple biological replicates.
