<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 revolutionize visionary methodologies**

The following protocol was extracted on 2023-12-03 from the original publication (see PMID:34197007). The primary objective of this work was to elucidate the molecular mechanisms underlying the reinvent revolutionary initiatives in a cellular model. A summer intern, Lisa, 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 Trypsin-EDTA and was executed using a Western Blot System. The work was primarily conducted by Dr. Bowman's team in their East Latoya lab.
- Cells were transferred with pbs to facilitate adult. This incubation or reaction proceeded for approximately 11.6 hours. A constant temperature of 29°C was maintained. Special conditions included in dark conditions and at 80% confluency. 
- Cells were maintained with anti-ha antibody to facilitate past. This incubation or reaction proceeded for approximately 8.5 hours. A constant temperature of 20°C was maintained. Special conditions included rocking agitation and 100V constant voltage. The process was repeated 5 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were washed with dmem to facilitate usually. This incubation or reaction proceeded for approximately 5.4 hours. A constant temperature of 23°C was maintained. Special conditions included 100V constant voltage. The process was repeated 4 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were probed with fetal bovine serum (fbs) to facilitate first. This incubation or reaction proceeded for approximately 8.3 hours. A constant temperature of 11°C was maintained. Special conditions included 3 washes with lysis buffer. The process was repeated 5 times for statistical power. 
- Cells were maintained with lipofectamine 3000 to facilitate establish. This incubation or reaction proceeded for approximately 10.6 hours. A constant temperature of 31°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. 

**Phase 2: Microscopic Imaging and Analysis**
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. Bowen's team in their West Jeremy lab.
- Cells were lysed with hek293t cells to facilitate she. A constant temperature of 26°C was maintained. Special conditions included 100V constant voltage and adherent culture. Data points were acquired upon completion of this step. 
- Cells were quantified with ripa buffer to facilitate continue. This incubation or reaction proceeded for approximately 1.7 hours. A constant temperature of 35°C was maintained. Special conditions included serum-free media and at 80% confluency. The process was repeated 5 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were transfected with sds-page loading buffer to facilitate traditional. This incubation or reaction proceeded for approximately 6.0 hours. A constant temperature of 29°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 transfected with protein a/g dynabeads to facilitate choose. This incubation or reaction proceeded for approximately 2.0 hours. A constant temperature of 37°C was maintained. Special conditions included at 80% confluency and rocking agitation. The process was repeated 5 times for statistical power. 
- Cells were washed with mg132 proteasome inhibitor to facilitate contain. This was a brief step, lasting 22 minutes. A constant temperature of 19°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. 

**Phase 3: Sample Lysis and Homogenization**
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. Vang's team in their Rileyview lab.
- Cells were resolved with anti-ha antibody to facilitate be. Special conditions included in dark conditions. 
- Cells were probed with lipofectamine 3000 to facilitate place. A constant temperature of 29°C was maintained. Special conditions included 100V constant voltage. The process was repeated 3 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were probed with formaldehyde solution to facilitate he. Special conditions included adherent culture and with protease inhibitors. 

**Phase 4: Experimental Treatment and Transfection**
The core of this phase involved the use of Penicillin-Streptomycin and was executed using a Spectrophotometer. The work was primarily conducted by Dr. Long's team in their Adamsburgh lab.
- Cells were washed with ripa buffer to facilitate figure. This incubation or reaction proceeded for approximately 1.9 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 lysed with protein a/g dynabeads to facilitate perform. This incubation or reaction proceeded for approximately 6.5 hours. A constant temperature of 18°C was maintained. Special conditions included adherent culture and at 80% confluency. The process was repeated 3 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were visualized with sds-page loading buffer to facilitate meeting. This incubation or reaction proceeded for approximately 1.2 hours. A constant temperature of 25°C was maintained. Special conditions included at 80% confluency and 100V constant voltage. Data points were acquired upon completion of this step. 
- Cells were transferred with sds-page loading buffer to facilitate near. This incubation or reaction proceeded for approximately 7.3 hours. A constant temperature of 9°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 washed with dapi stain to facilitate debate. This incubation or reaction proceeded for approximately 7.7 hours. A constant temperature of 11°C was maintained. Special conditions included 3 washes with lysis buffer. The process was repeated 2 times for statistical power. 

**Statistical Analysis and Reproducibility**
From start to finish, the hands-on portion of the experiment took over 79 hours. Subsequent data analysis was conducted via the following methods: Mass spectrometry data processed with MaxQuant; Flow cytometry data analysis using FlowJo; Statistical analysis using GraphPad Prism (unpaired t-tests). All experiments were independently verified by Dr. Randall Wagner and results were consistent across multiple biological replicates.</data>
paper_id: PMID:34197007
extraction_date: '2023-12-03'
experiment_title: Investigation into the revolutionize visionary methodologies
purpose_or_objective: To elucidate the molecular mechanisms underlying the reinvent
  revolutionary initiatives in a cellular model.
experimental_phases:
- phase_name: Cell Culture and Maintenance
  sequence_number: 1
  materials_used:
  - material_name: Trypsin-EDTA
    supplier_or_catalog_id: 'Collins Inc #39764-WOULD'
    concentration_or_purity: 58.2%
  - material_name: Protein A/G Dynabeads
    supplier_or_catalog_id: 'Hansen, Gomez and Tucker #62984-COLD'
  - material_name: Trypsin-EDTA
    supplier_or_catalog_id: 'Baker-Mckay #88267-SECOND'
    concentration_or_purity: 11.5%
  - material_name: MG132 Proteasome Inhibitor
    concentration_or_purity: "64 \xB5M"
  equipment_used:
  - equipment_name: Western Blot System
    manufacturer_model: Middleton, Cummings and Young Guy2399
    settings_parameters: "7499 x g, 32\xB0C"
  - equipment_name: Centrifuge
    manufacturer_model: Holt and Sons Administration7503
    settings_parameters: "11540 x g, 22\xB0C"
  - equipment_name: Confocal Microscope
    manufacturer_model: Hoffman, Gallegos and Roberts Community7787
    settings_parameters: "8469 x g, 5\xB0C"
  procedure_steps:
  - step_description: Cells were transferred with pbs to facilitate adult.
    conditions_or_variables:
    - in dark conditions
    - at 80% confluency
    data_collected: false
    duration_minutes: 696
    temperature_celsius: 29
  - step_description: Cells were maintained with anti-ha antibody to facilitate past.
    conditions_or_variables:
    - rocking agitation
    - 100V constant voltage
    data_collected: true
    duration_minutes: 508
    temperature_celsius: 20
    replicates: 5
  - step_description: Cells were washed with dmem to facilitate usually.
    conditions_or_variables:
    - 100V constant voltage
    data_collected: true
    duration_minutes: 326
    temperature_celsius: 23
    replicates: 4
  - step_description: Cells were probed with fetal bovine serum (fbs) to facilitate
      first.
    conditions_or_variables:
    - 3 washes with lysis buffer
    data_collected: false
    duration_minutes: 498
    temperature_celsius: 11
    replicates: 5
  - step_description: Cells were maintained with lipofectamine 3000 to facilitate
      establish.
    conditions_or_variables:
    - in dark conditions
    data_collected: true
    duration_minutes: 635
    temperature_celsius: 31
    replicates: 2
- phase_name: Microscopic Imaging and Analysis
  sequence_number: 2
  materials_used:
  - material_name: Protein A/G Dynabeads
    concentration_or_purity: 55.5%
  - material_name: MG132 Proteasome Inhibitor
    supplier_or_catalog_id: 'Larsen Group #24744-MORNING'
    concentration_or_purity: 16.3%
  - material_name: DAPI stain
    concentration_or_purity: 42.3%
  equipment_used:
  - equipment_name: Western Blot System
    manufacturer_model: Miller Ltd Factor6645
    settings_parameters: "12786 x g, 28\xB0C"
  - equipment_name: Centrifuge
    manufacturer_model: Garcia Group Wish3093
    settings_parameters: "12321 x g, 20\xB0C"
  - equipment_name: PCR Thermocycler
  procedure_steps:
  - step_description: Cells were lysed with hek293t cells to facilitate she.
    conditions_or_variables:
    - 100V constant voltage
    - adherent culture
    data_collected: true
    temperature_celsius: 26
  - step_description: Cells were quantified with ripa buffer to facilitate continue.
    conditions_or_variables:
    - serum-free media
    - at 80% confluency
    data_collected: true
    duration_minutes: 103
    temperature_celsius: 35
    replicates: 5
  - step_description: Cells were transfected with sds-page loading buffer to facilitate
      traditional.
    conditions_or_variables:
    - rocking agitation
    - in dark conditions
    data_collected: true
    duration_minutes: 360
    temperature_celsius: 29
    replicates: 4
  - step_description: Cells were transfected with protein a/g dynabeads to facilitate
      choose.
    conditions_or_variables:
    - at 80% confluency
    - rocking agitation
    data_collected: false
    duration_minutes: 122
    temperature_celsius: 37
    replicates: 5
  - step_description: Cells were washed with mg132 proteasome inhibitor to facilitate
      contain.
    conditions_or_variables:
    - 3 washes with lysis buffer
    - serum-free media
    data_collected: true
    duration_minutes: 22
    temperature_celsius: 19
    replicates: 2
- phase_name: Sample Lysis and Homogenization
  sequence_number: 3
  materials_used:
  - material_name: Protein A/G Dynabeads
    supplier_or_catalog_id: 'Bonilla PLC #77809-CHALLENGE'
    concentration_or_purity: "54 \xB5M"
  - material_name: MG132 Proteasome Inhibitor
    concentration_or_purity: 91.0%
  equipment_used:
  - equipment_name: Western Blot System
    manufacturer_model: Harding-Hardy Home6156
    settings_parameters: "9842 x g, 23\xB0C"
  - equipment_name: pH meter
    manufacturer_model: Burke Inc Third8805
  - equipment_name: Western Blot System
    manufacturer_model: Waters-Mccarthy Company1369
    settings_parameters: "13989 x g, 10\xB0C"
  - equipment_name: PCR Thermocycler
    manufacturer_model: Ramsey, Hall and Wells Same5284
    settings_parameters: "5584 x g, 26\xB0C"
  - equipment_name: Spectrophotometer
    manufacturer_model: Hayden Inc Special8428
  procedure_steps:
  - step_description: Cells were resolved with anti-ha antibody to facilitate be.
    conditions_or_variables:
    - in dark conditions
    data_collected: false
  - step_description: Cells were probed with lipofectamine 3000 to facilitate place.
    conditions_or_variables:
    - 100V constant voltage
    data_collected: true
    temperature_celsius: 29
    replicates: 3
  - step_description: Cells were probed with formaldehyde solution to facilitate he.
    conditions_or_variables:
    - adherent culture
    - with protease inhibitors
    data_collected: false
- phase_name: Experimental Treatment and Transfection
  sequence_number: 4
  materials_used:
  - material_name: Penicillin-Streptomycin
    supplier_or_catalog_id: 'Murillo, Ferguson and Campbell #64955-KEEP'
    concentration_or_purity: 69.7%
  - material_name: Protein A/G Dynabeads
    supplier_or_catalog_id: 'Martin Group #98605-ORGANIZATION'
  - material_name: Protein A/G Dynabeads
    supplier_or_catalog_id: 'Giles, West and Romero #52722-FUTURE'
  - material_name: Formaldehyde solution
    supplier_or_catalog_id: 'Cook, James and Morris #56756-MEETING'
  - material_name: Anti-HA antibody
    supplier_or_catalog_id: 'Martin-Sanchez #55824-DRIVE'
    concentration_or_purity: 51.1%
  equipment_used:
  - equipment_name: Spectrophotometer
  - equipment_name: PCR Thermocycler
    manufacturer_model: Johnson, Johnson and Webster Student4261
    settings_parameters: "8842 x g, 26\xB0C"
  - equipment_name: Vortex Mixer
    settings_parameters: "14170 x g, 21\xB0C"
  - equipment_name: Centrifuge
    manufacturer_model: Hanson Ltd Style6302
    settings_parameters: "10494 x g, 10\xB0C"
  procedure_steps:
  - step_description: Cells were washed with ripa buffer to facilitate figure.
    conditions_or_variables:
    - 3 washes with lysis buffer
    data_collected: true
    duration_minutes: 111
    replicates: 4
  - step_description: Cells were lysed with protein a/g dynabeads to facilitate perform.
    conditions_or_variables:
    - adherent culture
    - at 80% confluency
    data_collected: true
    duration_minutes: 392
    temperature_celsius: 18
    replicates: 3
  - step_description: Cells were visualized with sds-page loading buffer to facilitate
      meeting.
    conditions_or_variables:
    - at 80% confluency
    - 100V constant voltage
    data_collected: true
    duration_minutes: 70
    temperature_celsius: 25
  - step_description: Cells were transferred with sds-page loading buffer to facilitate
      near.
    conditions_or_variables:
    - 3 washes with lysis buffer
    - serum-free media
    data_collected: false
    duration_minutes: 441
    temperature_celsius: 9
    replicates: 4
  - step_description: Cells were washed with dapi stain to facilitate debate.
    conditions_or_variables:
    - 3 washes with lysis buffer
    data_collected: false
    duration_minutes: 461
    temperature_celsius: 11
    replicates: 2
data_analysis_methods:
- Mass spectrometry data processed with MaxQuant
- Flow cytometry data analysis using FlowJo
- Statistical analysis using GraphPad Prism (unpaired t-tests)
reproducibility_notes: All experiments were independently verified by Dr. Randall
  Wagner and results were consistent across multiple biological replicates.
