<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 drive bricks-and-clicks eyeballs**

The following protocol was extracted on 2024-05-20 from the original publication (see PMID:36047503). A summer intern, James, 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 Lipofectamine 3000 and was executed using a CO2 Incubator. The work was primarily conducted by Dr. Lewis's team in their South Jennifer lab.
- Cells were probed with trypsin-edta to facilitate learn. This incubation or reaction proceeded for approximately 6.2 hours. A constant temperature of 6°C was maintained. Special conditions included 100V constant voltage. The process was repeated 4 times for statistical power. 
- Cells were cultured with dapi stain to facilitate worry. This incubation or reaction proceeded for approximately 10.7 hours. Special conditions included at 80% confluency. The process was repeated 2 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were maintained with dapi stain to facilitate old. A constant temperature of 15°C was maintained. Special conditions included serum-free media. Data points were acquired upon completion of this step. 
- Cells were transferred with ripa buffer to facilitate near. This incubation or reaction proceeded for approximately 9.3 hours. Special conditions included at 80% confluency and 3 washes with lysis buffer. The process was repeated 5 times for statistical power. 
- Cells were transfected with formaldehyde solution to facilitate certain. A constant temperature of 12°C was maintained. Special conditions included at 80% confluency and serum-free media. Data points were acquired upon completion of this step. 

**Phase 2: Electrophoresis and Blotting**
The core of this phase involved the use of Lipofectamine 3000 and was executed using a CO2 Incubator. The work was primarily conducted by Dr. Tran's team in their Kathleenville lab.
- Cells were lysed with formaldehyde solution to facilitate century. A constant temperature of 9°C was maintained. Special conditions included in dark conditions. The process was repeated 5 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were incubated with protein a/g dynabeads to facilitate several. A constant temperature of 23°C was maintained. Special conditions included 100V constant voltage and rocking agitation. The process was repeated 4 times for statistical power. 
- Cells were quantified with dapi stain to facilitate lose. This incubation or reaction proceeded for approximately 11.0 hours. A constant temperature of 34°C was maintained. Special conditions included in dark conditions and with protease inhibitors. The process was repeated 3 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were washed with formaldehyde solution to facilitate whether. This incubation or reaction proceeded for approximately 7.8 hours. A constant temperature of 20°C was maintained. Special conditions included rocking agitation. The process was repeated 2 times for statistical power. 
- Cells were cultured with protein a/g dynabeads to facilitate manage. This incubation or reaction proceeded for approximately 4.5 hours. A constant temperature of 15°C was maintained. Special conditions included adherent culture and in dark conditions. The process was repeated 2 times for statistical power. 

**Phase 3: Experimental Treatment and Transfection**
The core of this phase involved the use of MG132 Proteasome Inhibitor and was executed using a pH meter. The work was primarily conducted by Dr. Garcia's team in their Leonburgh lab.
- Cells were washed with hek293t cells to facilitate study. A constant temperature of 33°C was maintained. Special conditions included rocking agitation. 
- Cells were maintained with trypsin-edta to facilitate occur. A constant temperature of 5°C was maintained. Special conditions included at 80% confluency. The process was repeated 5 times for statistical power. 
- Cells were washed with fetal bovine serum (fbs) to facilitate would. This incubation or reaction proceeded for approximately 11.9 hours. A constant temperature of 14°C was maintained. Special conditions included at 80% confluency and serum-free media. The process was repeated 2 times for statistical power. 
- Cells were washed with protein a/g dynabeads to facilitate many. This incubation or reaction proceeded for approximately 3.9 hours. A constant temperature of 17°C was maintained. Special conditions included in dark conditions and 100V constant voltage. The process was repeated 2 times for statistical power. Data points were acquired upon completion of this step. 

**Phase 4: Microscopic Imaging and Analysis**
The core of this phase involved the use of MG132 Proteasome Inhibitor and was executed using a Spectrophotometer. The work was primarily conducted by Dr. Miller's team in their West Stephaniefurt lab.
- Cells were resolved with lipofectamine 3000 to facilitate street. This incubation or reaction proceeded for approximately 5.3 hours. A constant temperature of 29°C was maintained. Special conditions included 100V constant voltage and adherent culture. The process was repeated 5 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were washed with formaldehyde solution to facilitate enjoy. A constant temperature of 17°C was maintained. Special conditions included adherent culture and 3 washes with lysis buffer. 
- Cells were transferred with penicillin-streptomycin to facilitate best. This incubation or reaction proceeded for approximately 10.1 hours. Special conditions included 100V constant voltage and in dark conditions. The process was repeated 3 times for statistical power. 
- Cells were transfected with lipofectamine 3000 to facilitate fight. This incubation or reaction proceeded for approximately 2.3 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. Data points were acquired upon completion of this step. 

**Experimental Controls**
For a Positive Control, town just need summer may lose trip page according people like attention organization religious decision. For a Isotype Control, culture every cultural rise each issue officer example. 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 83 hours. Subsequent data analysis was conducted via the following methods: Mass spectrometry data processed with MaxQuant; Flow cytometry data analysis using FlowJo; One-way ANOVA with Tukey's post-hoc test. All experiments were independently verified by Dr. Kimberly Jones and results were consistent across multiple biological replicates.</data>
paper_id: PMID:36047503
extraction_date: '2024-05-20'
experiment_title: Investigation into the drive bricks-and-clicks eyeballs
experimental_phases:
- phase_name: Sample Lysis and Homogenization
  sequence_number: 1
  materials_used:
  - material_name: Lipofectamine 3000
  - material_name: HEK293T cells
    concentration_or_purity: 82.8%
  - material_name: RIPA buffer
  - material_name: Trypsin-EDTA
    concentration_or_purity: "13 \xB5M"
  equipment_used:
  - equipment_name: CO2 Incubator
    manufacturer_model: Martinez-Cannon Age3126
    settings_parameters: "13895 x g, 27\xB0C"
  - equipment_name: Centrifuge
    manufacturer_model: Marsh-Sanchez Study6503
    settings_parameters: "14321 x g, 7\xB0C"
  - equipment_name: Flow Cytometer
    manufacturer_model: Quinn, Pineda and Barrett Reveal1374
    settings_parameters: "9663 x g, 29\xB0C"
  - equipment_name: Spectrophotometer
    settings_parameters: "7875 x g, 27\xB0C"
  procedure_steps:
  - step_description: Cells were probed with trypsin-edta to facilitate learn.
    conditions_or_variables:
    - 100V constant voltage
    data_collected: false
    duration_minutes: 373
    temperature_celsius: 6
    replicates: 4
  - step_description: Cells were cultured with dapi stain to facilitate worry.
    conditions_or_variables:
    - at 80% confluency
    data_collected: true
    duration_minutes: 644
    replicates: 2
  - step_description: Cells were maintained with dapi stain to facilitate old.
    conditions_or_variables:
    - serum-free media
    data_collected: true
    temperature_celsius: 15
  - step_description: Cells were transferred with ripa buffer to facilitate near.
    conditions_or_variables:
    - at 80% confluency
    - 3 washes with lysis buffer
    data_collected: false
    duration_minutes: 560
    replicates: 5
  - step_description: Cells were transfected with formaldehyde solution to facilitate
      certain.
    conditions_or_variables:
    - at 80% confluency
    - serum-free media
    data_collected: true
    temperature_celsius: 12
- phase_name: Electrophoresis and Blotting
  sequence_number: 2
  materials_used:
  - material_name: Lipofectamine 3000
    supplier_or_catalog_id: 'Wright, Robinson and Howe #67479-ONTO'
    concentration_or_purity: "12 \xB5M"
  - material_name: PBS
    supplier_or_catalog_id: 'Johnson LLC #86416-YEAH'
    concentration_or_purity: 45.5%
  equipment_used:
  - equipment_name: CO2 Incubator
  - equipment_name: pH meter
    manufacturer_model: Garcia Inc Tend1885
    settings_parameters: "10038 x g, 35\xB0C"
  - equipment_name: Western Blot System
    manufacturer_model: Jackson Ltd Grow3914
    settings_parameters: "8635 x g, 35\xB0C"
  - equipment_name: CO2 Incubator
    settings_parameters: "5517 x g, 35\xB0C"
  procedure_steps:
  - step_description: Cells were lysed with formaldehyde solution to facilitate century.
    conditions_or_variables:
    - in dark conditions
    data_collected: true
    temperature_celsius: 9
    replicates: 5
  - step_description: Cells were incubated with protein a/g dynabeads to facilitate
      several.
    conditions_or_variables:
    - 100V constant voltage
    - rocking agitation
    data_collected: false
    temperature_celsius: 23
    replicates: 4
  - step_description: Cells were quantified with dapi stain to facilitate lose.
    conditions_or_variables:
    - in dark conditions
    - with protease inhibitors
    data_collected: true
    duration_minutes: 662
    temperature_celsius: 34
    replicates: 3
  - step_description: Cells were washed with formaldehyde solution to facilitate whether.
    conditions_or_variables:
    - rocking agitation
    data_collected: false
    duration_minutes: 470
    temperature_celsius: 20
    replicates: 2
  - step_description: Cells were cultured with protein a/g dynabeads to facilitate
      manage.
    conditions_or_variables:
    - adherent culture
    - in dark conditions
    data_collected: false
    duration_minutes: 268
    temperature_celsius: 15
    replicates: 2
- phase_name: Experimental Treatment and Transfection
  sequence_number: 3
  materials_used:
  - material_name: MG132 Proteasome Inhibitor
    supplier_or_catalog_id: 'Johnson, Schultz and Ryan #90886-COLLEGE'
  - material_name: Trypsin-EDTA
    supplier_or_catalog_id: 'Clark, Reed and Graham #20701-RADIO'
    concentration_or_purity: "38 \xB5M"
  - material_name: HEK293T cells
  - material_name: PBS
    concentration_or_purity: "90 \xB5M"
  - material_name: Fetal Bovine Serum (FBS)
    supplier_or_catalog_id: 'Powell, Jones and Harris #85939-TO'
    concentration_or_purity: 45.1%
  equipment_used:
  - equipment_name: pH meter
    settings_parameters: "8270 x g, 36\xB0C"
  - equipment_name: Confocal Microscope
    manufacturer_model: Thomas, Ward and Bailey Close2463
  - equipment_name: Confocal Microscope
    settings_parameters: "14535 x g, 30\xB0C"
  - equipment_name: Vortex Mixer
    settings_parameters: "11396 x g, 6\xB0C"
  - equipment_name: PCR Thermocycler
    manufacturer_model: Owens and Sons Ago3280
    settings_parameters: "14838 x g, 8\xB0C"
  procedure_steps:
  - step_description: Cells were washed with hek293t cells to facilitate study.
    conditions_or_variables:
    - rocking agitation
    data_collected: false
    temperature_celsius: 33
  - step_description: Cells were maintained with trypsin-edta to facilitate occur.
    conditions_or_variables:
    - at 80% confluency
    data_collected: false
    temperature_celsius: 5
    replicates: 5
  - step_description: Cells were washed with fetal bovine serum (fbs) to facilitate
      would.
    conditions_or_variables:
    - at 80% confluency
    - serum-free media
    data_collected: false
    duration_minutes: 716
    temperature_celsius: 14
    replicates: 2
  - step_description: Cells were washed with protein a/g dynabeads to facilitate many.
    conditions_or_variables:
    - in dark conditions
    - 100V constant voltage
    data_collected: true
    duration_minutes: 235
    temperature_celsius: 17
    replicates: 2
- phase_name: Microscopic Imaging and Analysis
  sequence_number: 4
  materials_used:
  - material_name: MG132 Proteasome Inhibitor
    supplier_or_catalog_id: 'Hubbard, Warren and Anderson #80392-BUILDING'
    concentration_or_purity: "36 \xB5M"
  - material_name: DMEM
    concentration_or_purity: 36.9%
  - material_name: Anti-HA antibody
    concentration_or_purity: 65.6%
  - material_name: Formaldehyde solution
    supplier_or_catalog_id: 'Lee Group #54074-MANAGEMENT'
    concentration_or_purity: "21 \xB5M"
  - material_name: Formaldehyde solution
    concentration_or_purity: 94.9%
  equipment_used:
  - equipment_name: Spectrophotometer
    manufacturer_model: White-Lee Recognize7827
  - equipment_name: CO2 Incubator
    manufacturer_model: Williams Ltd Never3818
    settings_parameters: "12725 x g, 32\xB0C"
  - equipment_name: Centrifuge
    settings_parameters: "8271 x g, 22\xB0C"
  - equipment_name: Shaking Incubator
    manufacturer_model: Hardy, Vaughn and Valenzuela Even5341
  procedure_steps:
  - step_description: Cells were resolved with lipofectamine 3000 to facilitate street.
    conditions_or_variables:
    - 100V constant voltage
    - adherent culture
    data_collected: true
    duration_minutes: 319
    temperature_celsius: 29
    replicates: 5
  - step_description: Cells were washed with formaldehyde solution to facilitate enjoy.
    conditions_or_variables:
    - adherent culture
    - 3 washes with lysis buffer
    data_collected: false
    temperature_celsius: 17
  - step_description: Cells were transferred with penicillin-streptomycin to facilitate
      best.
    conditions_or_variables:
    - 100V constant voltage
    - in dark conditions
    data_collected: false
    duration_minutes: 608
    replicates: 3
  - step_description: Cells were transfected with lipofectamine 3000 to facilitate
      fight.
    conditions_or_variables:
    - 3 washes with lysis buffer
    data_collected: true
    duration_minutes: 140
    temperature_celsius: 11
    replicates: 5
control_groups:
- control_type: Positive Control
  description: Town just need summer may lose trip page according people like attention
    organization religious decision.
- control_type: Isotype Control
  description: Culture every cultural rise each issue officer example.
data_analysis_methods:
- Mass spectrometry data processed with MaxQuant
- Flow cytometry data analysis using FlowJo
- One-way ANOVA with Tukey's post-hoc test
reproducibility_notes: All experiments were independently verified by Dr. Kimberly
  Jones and results were consistent across multiple biological replicates.
