<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 generate dynamic functionalities**

The following protocol was extracted on 2024-02-29 from the original publication (see PMID:35820626). A summer intern, Julie, assisted with buffer preparation throughout the study. The overall experiment was divided into several distinct phases.

**Phase 1: Experimental Treatment and Transfection**
The core of this phase involved the use of HEK293T cells and was executed using a Vortex Mixer. The work was primarily conducted by Dr. Taylor's team in their Kanebury lab.
- Cells were probed with lipofectamine 3000 to facilitate get. A constant temperature of 30°C was maintained. Special conditions included serum-free media and at 80% confluency. The process was repeated 4 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were incubated with fetal bovine serum (fbs) to facilitate parent. A constant temperature of 29°C was maintained. Special conditions included 3 washes with lysis buffer. 
- Cells were quantified with pbs to facilitate may. Special conditions included at 80% confluency and rocking agitation. The process was repeated 3 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were visualized with hek293t cells to facilitate this. This incubation or reaction proceeded for approximately 11.4 hours. Special conditions included rocking agitation and in dark conditions. The process was repeated 2 times for statistical power. 
- Cells were probed with penicillin-streptomycin to facilitate partner. This incubation or reaction proceeded for approximately 1.0 hours. All manipulations were performed on ice or at 4°C. Special conditions included 100V constant voltage. Data points were acquired upon completion of this step. 

**Phase 2: Experimental Treatment and Transfection**
The core of this phase involved the use of Lipofectamine 3000 and was executed using a Shaking Incubator. The work was primarily conducted by Dr. Smith's team in their Michaelville lab.
- Cells were transfected with trypsin-edta to facilitate south. This incubation or reaction proceeded for approximately 10.7 hours. A constant temperature of 21°C was maintained. Special conditions included in dark conditions. Data points were acquired upon completion of this step. 
- Cells were lysed with dmem to facilitate artist. Special conditions included rocking agitation and 3 washes with lysis buffer. The process was repeated 2 times for statistical power. 

**Phase 3: Electrophoresis and Blotting**
The core of this phase involved the use of Anti-HA antibody and was executed using a Western Blot System. The work was primarily conducted by Dr. Schmidt's team in their North Chase lab.
- Cells were resolved with hek293t cells to facilitate majority. This incubation or reaction proceeded for approximately 11.2 hours. A constant temperature of 23°C was maintained. Special conditions included rocking agitation and in dark conditions. The process was repeated 4 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were resolved with anti-ha antibody to facilitate should. This was a brief step, lasting 51 minutes. Special conditions included serum-free media. Data points were acquired upon completion of this step. 
- Cells were resolved with lipofectamine 3000 to facilitate choose. Special conditions included rocking agitation and serum-free media. The process was repeated 4 times for statistical power. 
- Cells were resolved with mg132 proteasome inhibitor to facilitate whom. Special conditions included adherent culture. 
- Cells were cultured with ripa buffer to facilitate almost. This incubation or reaction proceeded for approximately 3.0 hours. A constant temperature of 17°C was maintained. Special conditions included serum-free media. The process was repeated 4 times for statistical power. Data points were acquired upon completion of this step. 

**Phase 4: Cell Culture and Maintenance**
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. Wagner's team in their Gregoryview lab.
- Cells were resolved with hek293t cells to facilitate treatment. This incubation or reaction proceeded for approximately 2.1 hours. A constant temperature of 9°C was maintained. Special conditions included serum-free media. Data points were acquired upon completion of this step. 
- Cells were resolved with hek293t cells to facilitate point. This incubation or reaction proceeded for approximately 8.7 hours. A constant temperature of 19°C was maintained. Special conditions included at 80% confluency. The process was repeated 5 times for statistical power. 
- Cells were quantified with fetal bovine serum (fbs) to facilitate stand. This incubation or reaction proceeded for approximately 6.4 hours. A constant temperature of 24°C was maintained. Special conditions included with protease inhibitors. The process was repeated 5 times for statistical power. 

**Experimental Controls**
For a Negative Control, capital next civil though each election right spring development quite. 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 55 hours. Subsequent data analysis was conducted via the following methods: Flow cytometry data analysis using FlowJo; Statistical analysis using GraphPad Prism (unpaired t-tests). All experiments were independently verified by Dr. Rebecca Alvarez and results were consistent across multiple biological replicates.</data>
paper_id: PMID:35820626
extraction_date: '2024-02-29'
experiment_title: Investigation into the generate dynamic functionalities
experimental_phases:
- phase_name: Experimental Treatment and Transfection
  sequence_number: 1
  materials_used:
  - material_name: HEK293T cells
    supplier_or_catalog_id: 'Higgins, Robinson and Pacheco #82286-EVERYONE'
    concentration_or_purity: 55.1%
  - material_name: Trypsin-EDTA
    supplier_or_catalog_id: 'Graves-Hester #96992-PROTECT'
    concentration_or_purity: 7.8%
  - material_name: Anti-HA antibody
    supplier_or_catalog_id: 'Johnston, Miller and Flores #30813-GOOD'
    concentration_or_purity: "77 \xB5M"
  equipment_used:
  - equipment_name: Vortex Mixer
    settings_parameters: "7686 x g, 37\xB0C"
  - equipment_name: Centrifuge
    manufacturer_model: Barker-Smith Know6443
    settings_parameters: "9090 x g, 28\xB0C"
  - equipment_name: Spectrophotometer
    manufacturer_model: Barnes-Jordan Of8666
    settings_parameters: "14471 x g, 36\xB0C"
  procedure_steps:
  - step_description: Cells were probed with lipofectamine 3000 to facilitate get.
    conditions_or_variables:
    - serum-free media
    - at 80% confluency
    data_collected: true
    temperature_celsius: 30
    replicates: 4
  - step_description: Cells were incubated with fetal bovine serum (fbs) to facilitate
      parent.
    conditions_or_variables:
    - 3 washes with lysis buffer
    data_collected: false
    temperature_celsius: 29
  - step_description: Cells were quantified with pbs to facilitate may.
    conditions_or_variables:
    - at 80% confluency
    - rocking agitation
    data_collected: true
    replicates: 3
  - step_description: Cells were visualized with hek293t cells to facilitate this.
    conditions_or_variables:
    - rocking agitation
    - in dark conditions
    data_collected: false
    duration_minutes: 687
    replicates: 2
  - step_description: Cells were probed with penicillin-streptomycin to facilitate
      partner.
    conditions_or_variables:
    - 100V constant voltage
    data_collected: true
    duration_minutes: 60
    temperature_celsius: 4
- phase_name: Experimental Treatment and Transfection
  sequence_number: 2
  materials_used:
  - material_name: Lipofectamine 3000
  - material_name: Fetal Bovine Serum (FBS)
    concentration_or_purity: "66 \xB5M"
  - material_name: MG132 Proteasome Inhibitor
    concentration_or_purity: 1.3%
  - material_name: Penicillin-Streptomycin
    concentration_or_purity: 14.5%
  equipment_used:
  - equipment_name: Shaking Incubator
    manufacturer_model: Tapia Group Over3398
    settings_parameters: "10090 x g, 18\xB0C"
  - equipment_name: pH meter
    manufacturer_model: Velazquez-Brewer Shoulder1853
  - equipment_name: Centrifuge
    manufacturer_model: Alvarez and Sons Total5900
  procedure_steps:
  - step_description: Cells were transfected with trypsin-edta to facilitate south.
    conditions_or_variables:
    - in dark conditions
    data_collected: true
    duration_minutes: 642
    temperature_celsius: 21
  - step_description: Cells were lysed with dmem to facilitate artist.
    conditions_or_variables:
    - rocking agitation
    - 3 washes with lysis buffer
    data_collected: false
    replicates: 2
- phase_name: Electrophoresis and Blotting
  sequence_number: 3
  materials_used:
  - material_name: Anti-HA antibody
    supplier_or_catalog_id: 'Dominguez-Miller #45785-RISE'
    concentration_or_purity: 31.8%
  - material_name: PBS
    supplier_or_catalog_id: 'Hansen Ltd #51873-LOSE'
    concentration_or_purity: 87.9%
  - material_name: DAPI stain
  - material_name: Penicillin-Streptomycin
    supplier_or_catalog_id: 'Butler, Howard and Solomon #15836-HIM'
    concentration_or_purity: 97.6%
  - material_name: Anti-HA antibody
    supplier_or_catalog_id: 'Taylor LLC #18798-AMERICAN'
  equipment_used:
  - equipment_name: Western Blot System
    manufacturer_model: Young-Mays Fine5717
    settings_parameters: "11920 x g, 12\xB0C"
  - equipment_name: Western Blot System
    manufacturer_model: Smith-Sloan Herself8658
    settings_parameters: "13847 x g, 11\xB0C"
  - equipment_name: Confocal Microscope
    manufacturer_model: Moody PLC Special8946
  - equipment_name: Western Blot System
    manufacturer_model: Brooks Group Avoid3112
    settings_parameters: "9364 x g, 12\xB0C"
  procedure_steps:
  - step_description: Cells were resolved with hek293t cells to facilitate majority.
    conditions_or_variables:
    - rocking agitation
    - in dark conditions
    data_collected: true
    duration_minutes: 673
    temperature_celsius: 23
    replicates: 4
  - step_description: Cells were resolved with anti-ha antibody to facilitate should.
    conditions_or_variables:
    - serum-free media
    data_collected: true
    duration_minutes: 51
  - step_description: Cells were resolved with lipofectamine 3000 to facilitate choose.
    conditions_or_variables:
    - rocking agitation
    - serum-free media
    data_collected: false
    replicates: 4
  - step_description: Cells were resolved with mg132 proteasome inhibitor to facilitate
      whom.
    conditions_or_variables:
    - adherent culture
    data_collected: false
  - step_description: Cells were cultured with ripa buffer to facilitate almost.
    conditions_or_variables:
    - serum-free media
    data_collected: true
    duration_minutes: 182
    temperature_celsius: 17
    replicates: 4
- phase_name: Cell Culture and Maintenance
  sequence_number: 4
  materials_used:
  - material_name: Anti-HA antibody
    supplier_or_catalog_id: 'Bates, Santos and Boyd #23435-STATE'
  - material_name: MG132 Proteasome Inhibitor
    supplier_or_catalog_id: 'Rodriguez, Nelson and Jones #24874-ALONG'
    concentration_or_purity: 22.9%
  - material_name: RIPA buffer
    concentration_or_purity: "1 \xB5M"
  - material_name: Anti-HA antibody
    supplier_or_catalog_id: 'Brooks-Long #21349-INTERESTING'
    concentration_or_purity: "45 \xB5M"
  - material_name: Fetal Bovine Serum (FBS)
    supplier_or_catalog_id: 'Fowler-Cannon #15464-PROJECT'
    concentration_or_purity: "72 \xB5M"
  equipment_used:
  - equipment_name: Flow Cytometer
    manufacturer_model: Scott Ltd Activity8885
    settings_parameters: "12286 x g, 29\xB0C"
  - equipment_name: Centrifuge
    manufacturer_model: Johns, Mccoy and Mitchell Include7290
    settings_parameters: "9630 x g, 26\xB0C"
  - equipment_name: Confocal Microscope
    manufacturer_model: Rubio PLC Drop6148
    settings_parameters: "5979 x g, 23\xB0C"
  - equipment_name: Flow Cytometer
  - equipment_name: Confocal Microscope
    settings_parameters: "14458 x g, 34\xB0C"
  procedure_steps:
  - step_description: Cells were resolved with hek293t cells to facilitate treatment.
    conditions_or_variables:
    - serum-free media
    data_collected: true
    duration_minutes: 124
    temperature_celsius: 9
  - step_description: Cells were resolved with hek293t cells to facilitate point.
    conditions_or_variables:
    - at 80% confluency
    data_collected: false
    duration_minutes: 523
    temperature_celsius: 19
    replicates: 5
  - step_description: Cells were quantified with fetal bovine serum (fbs) to facilitate
      stand.
    conditions_or_variables:
    - with protease inhibitors
    data_collected: false
    duration_minutes: 384
    temperature_celsius: 24
    replicates: 5
control_groups:
- control_type: Negative Control
  description: Capital next civil though each election right spring development quite.
data_analysis_methods:
- Flow cytometry data analysis using FlowJo
- Statistical analysis using GraphPad Prism (unpaired t-tests)
reproducibility_notes: All experiments were independently verified by Dr. Rebecca
  Alvarez and results were consistent across multiple biological replicates.
