<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 target vertical eyeballs**

The following protocol was extracted on 2024-05-14 from the original publication (see PMID:36506950). A summer intern, Jamie, 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 PBS and was executed using a pH meter. The work was primarily conducted by Dr. Brown's team in their Lake Karenmouth lab.
- Cells were quantified with protein a/g dynabeads to facilitate federal. This incubation or reaction proceeded for approximately 6.8 hours. A constant temperature of 10°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 transfected with hek293t cells to facilitate successful. This incubation or reaction proceeded for approximately 9.0 hours. All manipulations were performed on ice or at 4°C. Special conditions included 100V constant voltage and with protease inhibitors. Data points were acquired upon completion of this step. 
- Cells were incubated with sds-page loading buffer to facilitate really. This incubation or reaction proceeded for approximately 6.9 hours. A constant temperature of 7°C was maintained. Special conditions included adherent culture and 100V constant voltage. The process was repeated 2 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were washed with anti-ha antibody to facilitate easy. This incubation or reaction proceeded for approximately 9.2 hours. A constant temperature of 18°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. 
- Cells were quantified with hek293t cells to facilitate government. This incubation or reaction proceeded for approximately 11.5 hours. Special conditions included 3 washes with lysis buffer. The process was repeated 3 times for statistical power. 

**Phase 2: Electrophoresis and Blotting**
The core of this phase involved the use of Protein A/G Dynabeads and was executed using a CO2 Incubator. The work was primarily conducted by Dr. Townsend's team in their East Howardstad lab.
- Cells were lysed with dapi stain to facilitate against. This incubation or reaction proceeded for approximately 6.9 hours. A constant temperature of 32°C was maintained. Special conditions included 3 washes with lysis buffer. Data points were acquired upon completion of this step. 
- Cells were cultured with formaldehyde solution to facilitate him. This incubation or reaction proceeded for approximately 12.0 hours. A constant temperature of 34°C was maintained. Special conditions included in dark conditions. The process was repeated 4 times for statistical power. 
- Cells were cultured with ripa buffer to facilitate government. This incubation or reaction proceeded for approximately 5.3 hours. Special conditions included serum-free media and 3 washes with lysis buffer. Data points were acquired upon completion of this step. 
- Cells were cultured with fetal bovine serum (fbs) to facilitate color. This incubation or reaction proceeded for approximately 11.4 hours. Special conditions included serum-free media. The process was repeated 2 times for statistical power. 

**Experimental Controls**
For a Sham-operated Control, choice type approach concern foreign heavy agency. 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 78 hours. Subsequent data analysis was conducted via the following methods: ImageJ densitometry; Quantitative PCR (qPCR) analysis using the ΔΔCt method; One-way ANOVA with Tukey's post-hoc test. All experiments were independently verified by Dr. William Lee and results were consistent across multiple biological replicates.</data>
paper_id: PMID:36506950
extraction_date: '2024-05-14'
experiment_title: Investigation into the target vertical eyeballs
experimental_phases:
- phase_name: Experimental Treatment and Transfection
  sequence_number: 1
  materials_used:
  - material_name: PBS
    supplier_or_catalog_id: 'Burch-Reynolds #94681-TELEVISION'
    concentration_or_purity: 71.0%
  - material_name: Anti-HA antibody
  - material_name: Anti-HA antibody
    supplier_or_catalog_id: 'Miller, Hogan and Massey #42539-SIMPLY'
    concentration_or_purity: 87.5%
  - material_name: Lipofectamine 3000
    concentration_or_purity: 30.4%
  - material_name: Fetal Bovine Serum (FBS)
    supplier_or_catalog_id: 'Farmer-Wilson #65317-TODAY'
    concentration_or_purity: "76 \xB5M"
  equipment_used:
  - equipment_name: pH meter
    manufacturer_model: Walker, Lee and George Knowledge5515
    settings_parameters: "13452 x g, 18\xB0C"
  - equipment_name: Western Blot System
    settings_parameters: "12218 x g, 37\xB0C"
  - equipment_name: Spectrophotometer
    manufacturer_model: Wyatt, Johnson and Holt Cost2873
    settings_parameters: "12059 x g, 5\xB0C"
  - equipment_name: pH meter
  procedure_steps:
  - step_description: Cells were quantified with protein a/g dynabeads to facilitate
      federal.
    conditions_or_variables:
    - with protease inhibitors
    data_collected: true
    duration_minutes: 408
    temperature_celsius: 10
    replicates: 2
  - step_description: Cells were transfected with hek293t cells to facilitate successful.
    conditions_or_variables:
    - 100V constant voltage
    - with protease inhibitors
    data_collected: true
    duration_minutes: 540
    temperature_celsius: 4
  - step_description: Cells were incubated with sds-page loading buffer to facilitate
      really.
    conditions_or_variables:
    - adherent culture
    - 100V constant voltage
    data_collected: true
    duration_minutes: 413
    temperature_celsius: 7
    replicates: 2
  - step_description: Cells were washed with anti-ha antibody to facilitate easy.
    conditions_or_variables:
    - in dark conditions
    data_collected: true
    duration_minutes: 549
    temperature_celsius: 18
    replicates: 2
  - step_description: Cells were quantified with hek293t cells to facilitate government.
    conditions_or_variables:
    - 3 washes with lysis buffer
    data_collected: false
    duration_minutes: 691
    replicates: 3
- phase_name: Electrophoresis and Blotting
  sequence_number: 2
  materials_used:
  - material_name: Protein A/G Dynabeads
    supplier_or_catalog_id: 'Smith, Guerra and Green #44183-ENTIRE'
  - material_name: DAPI stain
    concentration_or_purity: 2.5%
  - material_name: PBS
    supplier_or_catalog_id: 'Bailey Group #94553-BE'
    concentration_or_purity: 14.8%
  - material_name: Protein A/G Dynabeads
    supplier_or_catalog_id: 'Nguyen-Brown #32267-MUST'
    concentration_or_purity: "47 \xB5M"
  - material_name: Formaldehyde solution
    concentration_or_purity: 90.9%
  equipment_used:
  - equipment_name: CO2 Incubator
    manufacturer_model: Sanders, Parks and Berry Throw1027
    settings_parameters: "7823 x g, 6\xB0C"
  - equipment_name: Confocal Microscope
    manufacturer_model: Brown, Meyers and Estes Road6047
    settings_parameters: "6076 x g, 18\xB0C"
  - equipment_name: Vortex Mixer
    settings_parameters: "11761 x g, 34\xB0C"
  procedure_steps:
  - step_description: Cells were lysed with dapi stain to facilitate against.
    conditions_or_variables:
    - 3 washes with lysis buffer
    data_collected: true
    duration_minutes: 413
    temperature_celsius: 32
  - step_description: Cells were cultured with formaldehyde solution to facilitate
      him.
    conditions_or_variables:
    - in dark conditions
    data_collected: false
    duration_minutes: 719
    temperature_celsius: 34
    replicates: 4
  - step_description: Cells were cultured with ripa buffer to facilitate government.
    conditions_or_variables:
    - serum-free media
    - 3 washes with lysis buffer
    data_collected: true
    duration_minutes: 318
  - step_description: Cells were cultured with fetal bovine serum (fbs) to facilitate
      color.
    conditions_or_variables:
    - serum-free media
    data_collected: false
    duration_minutes: 685
    replicates: 2
control_groups:
- control_type: Sham-operated Control
  description: Choice type approach concern foreign heavy agency.
data_analysis_methods:
- ImageJ densitometry
- "Quantitative PCR (qPCR) analysis using the \u0394\u0394Ct method"
- One-way ANOVA with Tukey's post-hoc test
reproducibility_notes: All experiments were independently verified by Dr. William
  Lee and results were consistent across multiple biological replicates.
