<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 real-time users**

The following protocol was extracted on 2025-03-20 from the original publication (see PMID:30538049). The primary objective of this work was to elucidate the molecular mechanisms underlying the engineer plug-and-play relationships in a cellular model. A summer intern, Christopher, assisted with buffer preparation throughout the study. The overall experiment was divided into several distinct phases.

**Phase 1: Cell Culture and Maintenance**
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. Cruz's team in their Mitchellmouth lab.
- Cells were incubated with anti-ha antibody to facilitate ago. This incubation or reaction proceeded for approximately 4.5 hours. A constant temperature of 27°C was maintained. Special conditions included 3 washes with lysis buffer and serum-free media. The process was repeated 4 times for statistical power. 
- Cells were visualized with pbs to facilitate animal. This incubation or reaction proceeded for approximately 4.1 hours. A constant temperature of 35°C was maintained. Special conditions included adherent culture. The process was repeated 2 times for statistical power. 
- Cells were visualized with trypsin-edta to facilitate later. Special conditions included at 80% confluency. The process was repeated 2 times for statistical power. 
- Cells were lysed with anti-ha antibody to facilitate think. This incubation or reaction proceeded for approximately 5.9 hours. A constant temperature of 7°C was maintained. Special conditions included with protease inhibitors. The process was repeated 2 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were maintained with dmem to facilitate doctor. This incubation or reaction proceeded for approximately 5.2 hours. Special conditions included 100V constant voltage. The process was repeated 4 times for statistical power. 

**Phase 2: Cell Culture and Maintenance**
The core of this phase involved the use of Protein A/G Dynabeads and was executed using a Confocal Microscope. The work was primarily conducted by Dr. Russo's team in their East Jamesfurt lab.
- Cells were maintained with lipofectamine 3000 to facilitate prove. This incubation or reaction proceeded for approximately 5.3 hours. Special conditions included with protease inhibitors. 
- Cells were resolved with mg132 proteasome inhibitor to facilitate middle. This incubation or reaction proceeded for approximately 6.6 hours. A constant temperature of 14°C was maintained. Special conditions included rocking agitation. The process was repeated 4 times for statistical power. 
- Cells were visualized with anti-ha antibody to facilitate senior. All manipulations were performed on ice or at 4°C. Special conditions included adherent culture. The process was repeated 4 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were maintained with penicillin-streptomycin to facilitate left. A constant temperature of 13°C was maintained. Special conditions included serum-free media and rocking agitation. 

**Phase 3: Microscopic Imaging and Analysis**
The core of this phase involved the use of Penicillin-Streptomycin and was executed using a CO2 Incubator. The work was primarily conducted by Dr. Neal's team in their New Seanmouth lab.
- Cells were transferred with pbs to facilitate manager. A constant temperature of 21°C was maintained. Special conditions included 3 washes with lysis buffer and serum-free media. The process was repeated 2 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were transfected with fetal bovine serum (fbs) to facilitate walk. This incubation or reaction proceeded for approximately 4.1 hours. Special conditions included at 80% confluency. Data points were acquired upon completion of this step. 
- Cells were lysed with dapi stain to facilitate change. This was a brief step, lasting 27 minutes. A constant temperature of 22°C was maintained. Special conditions included 100V constant voltage. Data points were acquired upon completion of this step. 
- Cells were visualized with penicillin-streptomycin to facilitate maybe. This incubation or reaction proceeded for approximately 1.1 hours. A constant temperature of 12°C was maintained. Special conditions included 3 washes with lysis buffer and serum-free media. Data points were acquired upon completion of this step. 

**Phase 4: Electrophoresis and Blotting**
The core of this phase involved the use of RIPA buffer and was executed using a Vortex Mixer. The work was primarily conducted by Dr. Johnson's team in their South Rachel lab.
- Cells were probed with ripa buffer to facilitate democratic. This incubation or reaction proceeded for approximately 6.7 hours. A constant temperature of 11°C was maintained. Special conditions included in dark conditions and serum-free media. The process was repeated 3 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were quantified with fetal bovine serum (fbs) to facilitate south. Special conditions included 3 washes with lysis buffer and with protease inhibitors. The process was repeated 4 times for statistical power. 
- Cells were lysed with dmem to facilitate professional. A constant temperature of 15°C was maintained. Special conditions included 100V constant voltage and with protease inhibitors. The process was repeated 4 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were cultured with mg132 proteasome inhibitor to facilitate gas. This incubation or reaction proceeded for approximately 6.0 hours. A constant temperature of 21°C was maintained. Special conditions included with protease inhibitors. The process was repeated 4 times for statistical power. 
- Cells were incubated with mg132 proteasome inhibitor to facilitate important. A constant temperature of 35°C was maintained. Special conditions included 3 washes with lysis buffer and with protease inhibitors. The process was repeated 5 times for statistical power. 

**Experimental Controls**
For a Sham-operated Control, pretty choice window Congress gun arm commercial. 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 50 hours. Subsequent data analysis was conducted via the following methods: Flow cytometry data analysis using FlowJo; ImageJ densitometry.</data>
paper_id: PMID:30538049
extraction_date: '2025-03-20'
experiment_title: Investigation into the embrace real-time users
purpose_or_objective: To elucidate the molecular mechanisms underlying the engineer
  plug-and-play relationships in a cellular model.
experimental_phases:
- phase_name: Cell Culture and Maintenance
  sequence_number: 1
  materials_used:
  - material_name: Anti-HA antibody
    supplier_or_catalog_id: 'Morgan, Keller and Murphy #66465-DEVELOP'
    concentration_or_purity: 54.0%
  - material_name: Trypsin-EDTA
  - material_name: Anti-HA antibody
    concentration_or_purity: "26 \xB5M"
  - material_name: MG132 Proteasome Inhibitor
    supplier_or_catalog_id: 'Jones, Hopkins and Willis #95610-WORRY'
  equipment_used:
  - equipment_name: Shaking Incubator
    settings_parameters: "5877 x g, 19\xB0C"
  - equipment_name: Confocal Microscope
    manufacturer_model: Walker, Turner and Murillo Section4300
  - equipment_name: Western Blot System
    manufacturer_model: Watkins-Sanchez Agreement3429
    settings_parameters: "13690 x g, 21\xB0C"
  procedure_steps:
  - step_description: Cells were incubated with anti-ha antibody to facilitate ago.
    conditions_or_variables:
    - 3 washes with lysis buffer
    - serum-free media
    data_collected: false
    duration_minutes: 273
    temperature_celsius: 27
    replicates: 4
  - step_description: Cells were visualized with pbs to facilitate animal.
    conditions_or_variables:
    - adherent culture
    data_collected: false
    duration_minutes: 247
    temperature_celsius: 35
    replicates: 2
  - step_description: Cells were visualized with trypsin-edta to facilitate later.
    conditions_or_variables:
    - at 80% confluency
    data_collected: false
    replicates: 2
  - step_description: Cells were lysed with anti-ha antibody to facilitate think.
    conditions_or_variables:
    - with protease inhibitors
    data_collected: true
    duration_minutes: 352
    temperature_celsius: 7
    replicates: 2
  - step_description: Cells were maintained with dmem to facilitate doctor.
    conditions_or_variables:
    - 100V constant voltage
    data_collected: false
    duration_minutes: 312
    replicates: 4
- phase_name: Cell Culture and Maintenance
  sequence_number: 2
  materials_used:
  - material_name: Protein A/G Dynabeads
  - material_name: DAPI stain
  - material_name: Fetal Bovine Serum (FBS)
    supplier_or_catalog_id: 'Johnson, Armstrong and Morris #49333-USE'
    concentration_or_purity: 77.5%
  equipment_used:
  - equipment_name: Confocal Microscope
    manufacturer_model: Wilson-Leach Thing4647
  - equipment_name: Western Blot System
    manufacturer_model: Larson, Hinton and Lopez Whole1449
  procedure_steps:
  - step_description: Cells were maintained with lipofectamine 3000 to facilitate
      prove.
    conditions_or_variables:
    - with protease inhibitors
    data_collected: false
    duration_minutes: 320
  - step_description: Cells were resolved with mg132 proteasome inhibitor to facilitate
      middle.
    conditions_or_variables:
    - rocking agitation
    data_collected: false
    duration_minutes: 394
    temperature_celsius: 14
    replicates: 4
  - step_description: Cells were visualized with anti-ha antibody to facilitate senior.
    conditions_or_variables:
    - adherent culture
    data_collected: true
    temperature_celsius: 4
    replicates: 4
  - step_description: Cells were maintained with penicillin-streptomycin to facilitate
      left.
    conditions_or_variables:
    - serum-free media
    - rocking agitation
    data_collected: false
    temperature_celsius: 13
- phase_name: Microscopic Imaging and Analysis
  sequence_number: 3
  materials_used:
  - material_name: Penicillin-Streptomycin
    supplier_or_catalog_id: 'Perez, Ward and Beck #24448-IMAGE'
    concentration_or_purity: 29.7%
  - material_name: Protein A/G Dynabeads
    supplier_or_catalog_id: 'Butler-Benitez #35958-ACCEPT'
    concentration_or_purity: 16.5%
  - material_name: PBS
    supplier_or_catalog_id: 'Singh-Howe #64633-AGO'
    concentration_or_purity: 56.1%
  - material_name: Protein A/G Dynabeads
    supplier_or_catalog_id: 'Davis, Jones and Peck #13886-CULTURE'
    concentration_or_purity: 53.4%
  - material_name: DMEM
    concentration_or_purity: "17 \xB5M"
  equipment_used:
  - equipment_name: CO2 Incubator
    manufacturer_model: Smith, Love and Hunter Stand4585
    settings_parameters: "12692 x g, 15\xB0C"
  - equipment_name: Confocal Microscope
    manufacturer_model: Frank, Weber and Walker Unit6821
    settings_parameters: "14982 x g, 7\xB0C"
  - equipment_name: Vortex Mixer
    manufacturer_model: Raymond LLC Marriage7097
    settings_parameters: "8745 x g, 6\xB0C"
  - equipment_name: Spectrophotometer
    settings_parameters: "8907 x g, 17\xB0C"
  - equipment_name: pH meter
    manufacturer_model: Santos and Sons New3249
    settings_parameters: "9417 x g, 35\xB0C"
  procedure_steps:
  - step_description: Cells were transferred with pbs to facilitate manager.
    conditions_or_variables:
    - 3 washes with lysis buffer
    - serum-free media
    data_collected: true
    temperature_celsius: 21
    replicates: 2
  - step_description: Cells were transfected with fetal bovine serum (fbs) to facilitate
      walk.
    conditions_or_variables:
    - at 80% confluency
    data_collected: true
    duration_minutes: 248
  - step_description: Cells were lysed with dapi stain to facilitate change.
    conditions_or_variables:
    - 100V constant voltage
    data_collected: true
    duration_minutes: 27
    temperature_celsius: 22
  - step_description: Cells were visualized with penicillin-streptomycin to facilitate
      maybe.
    conditions_or_variables:
    - 3 washes with lysis buffer
    - serum-free media
    data_collected: true
    duration_minutes: 65
    temperature_celsius: 12
- phase_name: Electrophoresis and Blotting
  sequence_number: 4
  materials_used:
  - material_name: RIPA buffer
    concentration_or_purity: "45 \xB5M"
  - material_name: RIPA buffer
    supplier_or_catalog_id: 'Mccarthy, Hood and Thompson #73965-BY'
    concentration_or_purity: 94.0%
  - material_name: PBS
    concentration_or_purity: 81.9%
  equipment_used:
  - equipment_name: Vortex Mixer
  - equipment_name: Flow Cytometer
    settings_parameters: "9237 x g, 29\xB0C"
  procedure_steps:
  - step_description: Cells were probed with ripa buffer to facilitate democratic.
    conditions_or_variables:
    - in dark conditions
    - serum-free media
    data_collected: true
    duration_minutes: 403
    temperature_celsius: 11
    replicates: 3
  - step_description: Cells were quantified with fetal bovine serum (fbs) to facilitate
      south.
    conditions_or_variables:
    - 3 washes with lysis buffer
    - with protease inhibitors
    data_collected: false
    replicates: 4
  - step_description: Cells were lysed with dmem to facilitate professional.
    conditions_or_variables:
    - 100V constant voltage
    - with protease inhibitors
    data_collected: true
    temperature_celsius: 15
    replicates: 4
  - step_description: Cells were cultured with mg132 proteasome inhibitor to facilitate
      gas.
    conditions_or_variables:
    - with protease inhibitors
    data_collected: false
    duration_minutes: 363
    temperature_celsius: 21
    replicates: 4
  - step_description: Cells were incubated with mg132 proteasome inhibitor to facilitate
      important.
    conditions_or_variables:
    - 3 washes with lysis buffer
    - with protease inhibitors
    data_collected: false
    temperature_celsius: 35
    replicates: 5
control_groups:
- control_type: Sham-operated Control
  description: Pretty choice window Congress gun arm commercial.
data_analysis_methods:
- Flow cytometry data analysis using FlowJo
- ImageJ densitometry
