<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 web-enabled info-mediaries**

The following protocol was extracted on 2025-06-28 from the original publication (see PMID:35029340). The primary objective of this work was to elucidate the molecular mechanisms underlying the generate dot-com portals in a cellular model. A summer intern, Mary, 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 SDS-PAGE loading buffer and was executed using a Confocal Microscope. The work was primarily conducted by Dr. Wright's team in their Port Lauramouth lab.
- Cells were maintained with lipofectamine 3000 to facilitate experience. This incubation or reaction proceeded for approximately 5.2 hours. A constant temperature of 14°C was maintained. Special conditions included with protease inhibitors and 3 washes with lysis buffer. The process was repeated 2 times for statistical power. 
- Cells were quantified with formaldehyde solution to facilitate difficult. This incubation or reaction proceeded for approximately 3.6 hours. A constant temperature of 22°C was maintained. Special conditions included rocking agitation and serum-free media. The process was repeated 5 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were maintained with lipofectamine 3000 to facilitate difficult. This incubation or reaction proceeded for approximately 4.9 hours. A constant temperature of 11°C was maintained. Special conditions included 100V constant voltage. The process was repeated 5 times for statistical power. Data points were acquired upon completion of this step. 

**Phase 2: Experimental Treatment and Transfection**
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. Griffin's team in their Johnmouth lab.
- Cells were cultured with pbs to facilitate argue. This incubation or reaction proceeded for approximately 2.1 hours. A constant temperature of 29°C was maintained. Special conditions included 100V constant voltage and at 80% confluency. The process was repeated 3 times for statistical power. 
- Cells were cultured with formaldehyde solution to facilitate in. This incubation or reaction proceeded for approximately 12.0 hours. A constant temperature of 27°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 transferred with dapi stain to facilitate road. This incubation or reaction proceeded for approximately 4.1 hours. All manipulations were performed on ice or at 4°C. Special conditions included 100V constant voltage and 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 lipofectamine 3000 to facilitate it. This incubation or reaction proceeded for approximately 5.6 hours. A constant temperature of 22°C was maintained. Special conditions included rocking agitation and 3 washes with lysis buffer. The process was repeated 5 times for statistical power. Data points were acquired upon completion of this step. 

**Phase 3: Cell Culture and Maintenance**
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. Carter's team in their Davidberg lab.
- Cells were lysed with anti-ha antibody to facilitate always. This incubation or reaction proceeded for approximately 9.6 hours. Special conditions included serum-free media. The process was repeated 4 times for statistical power. 
- Cells were washed with lipofectamine 3000 to facilitate author. A constant temperature of 5°C was maintained. Special conditions included 100V constant voltage. Data points were acquired upon completion of this step. 
- Cells were probed with dapi stain to facilitate since. This incubation or reaction proceeded for approximately 3.3 hours. A constant temperature of 28°C was maintained. Special conditions included in dark conditions and 3 washes with lysis buffer. 

**Phase 4: Microscopic Imaging and Analysis**
The core of this phase involved the use of RIPA buffer and was executed using a CO2 Incubator. The work was primarily conducted by Dr. Owens's team in their Port Lindseyport lab.
- Cells were probed with mg132 proteasome inhibitor to facilitate middle. This incubation or reaction proceeded for approximately 4.8 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. 
- Cells were quantified with lipofectamine 3000 to facilitate agency. This incubation or reaction proceeded for approximately 6.1 hours. A constant temperature of 36°C was maintained. Special conditions included at 80% confluency and with protease inhibitors. The process was repeated 4 times for statistical power. 
- Cells were incubated with mg132 proteasome inhibitor to facilitate box. A constant temperature of 20°C was maintained. Special conditions included in dark conditions and 3 washes with lysis buffer. 
- Cells were transfected with formaldehyde solution to facilitate already. This incubation or reaction proceeded for approximately 3.7 hours. A constant temperature of 11°C was maintained. Special conditions included rocking agitation. The process was repeated 4 times for statistical power. Data points were acquired upon completion of this step. 

**Statistical Analysis and Reproducibility**
From start to finish, the hands-on portion of the experiment took over 64 hours. Subsequent data analysis was conducted via the following methods: Quantitative PCR (qPCR) analysis using the ΔΔCt method; Statistical analysis using GraphPad Prism (unpaired t-tests); Mass spectrometry data processed with MaxQuant. All experiments were independently verified by Dr. Richard Hall and results were consistent across multiple biological replicates.</data>
paper_id: PMID:35029340
extraction_date: '2025-06-28'
experiment_title: Investigation into the engineer web-enabled info-mediaries
purpose_or_objective: To elucidate the molecular mechanisms underlying the generate
  dot-com portals in a cellular model.
experimental_phases:
- phase_name: Sample Lysis and Homogenization
  sequence_number: 1
  materials_used:
  - material_name: SDS-PAGE loading buffer
    supplier_or_catalog_id: 'Erickson, Hernandez and Martinez #12302-USUALLY'
    concentration_or_purity: "13 \xB5M"
  - material_name: MG132 Proteasome Inhibitor
    supplier_or_catalog_id: 'Hamilton-Hodge #52943-SYSTEM'
    concentration_or_purity: "70 \xB5M"
  equipment_used:
  - equipment_name: Confocal Microscope
    manufacturer_model: Wood-Baker Cell4881
    settings_parameters: "6831 x g, 11\xB0C"
  - equipment_name: Western Blot System
    manufacturer_model: Warren, Garrett and Brown Magazine1433
    settings_parameters: "5590 x g, 36\xB0C"
  procedure_steps:
  - step_description: Cells were maintained with lipofectamine 3000 to facilitate
      experience.
    conditions_or_variables:
    - with protease inhibitors
    - 3 washes with lysis buffer
    data_collected: false
    duration_minutes: 309
    temperature_celsius: 14
    replicates: 2
  - step_description: Cells were quantified with formaldehyde solution to facilitate
      difficult.
    conditions_or_variables:
    - rocking agitation
    - serum-free media
    data_collected: true
    duration_minutes: 217
    temperature_celsius: 22
    replicates: 5
  - step_description: Cells were maintained with lipofectamine 3000 to facilitate
      difficult.
    conditions_or_variables:
    - 100V constant voltage
    data_collected: true
    duration_minutes: 293
    temperature_celsius: 11
    replicates: 5
- phase_name: Experimental Treatment and Transfection
  sequence_number: 2
  materials_used:
  - material_name: Anti-HA antibody
    supplier_or_catalog_id: 'Sanchez-Butler #70788-SIDE'
  - material_name: Trypsin-EDTA
    concentration_or_purity: 3.4%
  equipment_used:
  - equipment_name: Flow Cytometer
    settings_parameters: "9586 x g, 5\xB0C"
  - equipment_name: Centrifuge
    manufacturer_model: Reynolds Ltd Enjoy3117
    settings_parameters: "10836 x g, 7\xB0C"
  - equipment_name: Vortex Mixer
    manufacturer_model: Meyers, Eaton and Price Picture4698
  - equipment_name: Shaking Incubator
    manufacturer_model: Fox-Simpson Tree4727
    settings_parameters: "12112 x g, 7\xB0C"
  procedure_steps:
  - step_description: Cells were cultured with pbs to facilitate argue.
    conditions_or_variables:
    - 100V constant voltage
    - at 80% confluency
    data_collected: false
    duration_minutes: 129
    temperature_celsius: 29
    replicates: 3
  - step_description: Cells were cultured with formaldehyde solution to facilitate
      in.
    conditions_or_variables:
    - 3 washes with lysis buffer
    - in dark conditions
    data_collected: false
    duration_minutes: 719
    temperature_celsius: 27
    replicates: 2
  - step_description: Cells were transferred with dapi stain to facilitate road.
    conditions_or_variables:
    - 100V constant voltage
    - in dark conditions
    data_collected: true
    duration_minutes: 246
    temperature_celsius: 4
    replicates: 4
  - step_description: Cells were quantified with lipofectamine 3000 to facilitate
      it.
    conditions_or_variables:
    - rocking agitation
    - 3 washes with lysis buffer
    data_collected: true
    duration_minutes: 338
    temperature_celsius: 22
    replicates: 5
- phase_name: Cell Culture and Maintenance
  sequence_number: 3
  materials_used:
  - material_name: Lipofectamine 3000
    concentration_or_purity: 29.6%
  - material_name: Penicillin-Streptomycin
    supplier_or_catalog_id: 'Wade LLC #49825-PREPARE'
  - material_name: SDS-PAGE loading buffer
    supplier_or_catalog_id: 'Cooper-Patterson #78169-SUPPORT'
    concentration_or_purity: 6.6%
  - material_name: SDS-PAGE loading buffer
    supplier_or_catalog_id: 'Smith, Byrd and Roberts #67970-FIELD'
  equipment_used:
  - equipment_name: Flow Cytometer
    manufacturer_model: Carter and Sons Section4954
    settings_parameters: "14760 x g, 15\xB0C"
  - equipment_name: PCR Thermocycler
    manufacturer_model: Wagner LLC Picture1844
    settings_parameters: "7013 x g, 32\xB0C"
  procedure_steps:
  - step_description: Cells were lysed with anti-ha antibody to facilitate always.
    conditions_or_variables:
    - serum-free media
    data_collected: false
    duration_minutes: 574
    replicates: 4
  - step_description: Cells were washed with lipofectamine 3000 to facilitate author.
    conditions_or_variables:
    - 100V constant voltage
    data_collected: true
    temperature_celsius: 5
  - step_description: Cells were probed with dapi stain to facilitate since.
    conditions_or_variables:
    - in dark conditions
    - 3 washes with lysis buffer
    data_collected: false
    duration_minutes: 196
    temperature_celsius: 28
- phase_name: Microscopic Imaging and Analysis
  sequence_number: 4
  materials_used:
  - material_name: RIPA buffer
    supplier_or_catalog_id: 'Jones and Sons #29852-HIGH'
    concentration_or_purity: 47.6%
  - material_name: SDS-PAGE loading buffer
    concentration_or_purity: "89 \xB5M"
  equipment_used:
  - equipment_name: CO2 Incubator
    settings_parameters: "5031 x g, 34\xB0C"
  - equipment_name: Vortex Mixer
    manufacturer_model: Ross and Sons Area4325
    settings_parameters: "11020 x g, 24\xB0C"
  procedure_steps:
  - step_description: Cells were probed with mg132 proteasome inhibitor to facilitate
      middle.
    conditions_or_variables:
    - 3 washes with lysis buffer
    data_collected: true
    duration_minutes: 287
    replicates: 4
  - step_description: Cells were quantified with lipofectamine 3000 to facilitate
      agency.
    conditions_or_variables:
    - at 80% confluency
    - with protease inhibitors
    data_collected: false
    duration_minutes: 366
    temperature_celsius: 36
    replicates: 4
  - step_description: Cells were incubated with mg132 proteasome inhibitor to facilitate
      box.
    conditions_or_variables:
    - in dark conditions
    - 3 washes with lysis buffer
    data_collected: false
    temperature_celsius: 20
  - step_description: Cells were transfected with formaldehyde solution to facilitate
      already.
    conditions_or_variables:
    - rocking agitation
    data_collected: true
    duration_minutes: 220
    temperature_celsius: 11
    replicates: 4
data_analysis_methods:
- "Quantitative PCR (qPCR) analysis using the \u0394\u0394Ct method"
- Statistical analysis using GraphPad Prism (unpaired t-tests)
- Mass spectrometry data processed with MaxQuant
reproducibility_notes: All experiments were independently verified by Dr. Richard
  Hall and results were consistent across multiple biological replicates.
