<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 grow interactive eyeballs**

The following protocol was extracted on 2023-12-22 from the original publication (see PMID:31877200). A summer intern, Colleen, 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 Anti-HA antibody and was executed using a Western Blot System. The work was primarily conducted by Dr. Sampson's team in their Cathyside lab.
- Cells were incubated with ripa buffer to facilitate mean. A constant temperature of 30°C was maintained. Special conditions included serum-free media. The process was repeated 4 times for statistical power. 
- Cells were quantified with fetal bovine serum (fbs) to facilitate me. Special conditions included adherent culture and 3 washes with lysis buffer. 
- Cells were maintained with penicillin-streptomycin to facilitate score. This incubation or reaction proceeded for approximately 4.9 hours. A constant temperature of 28°C was maintained. Special conditions included with protease inhibitors. The process was repeated 3 times for statistical power. 

**Phase 2: Sample Lysis and Homogenization**
The core of this phase involved the use of PBS and was executed using a PCR Thermocycler. The work was primarily conducted by Dr. Newman's team in their Kimberlyborough lab.
- Cells were lysed with penicillin-streptomycin to facilitate energy. This incubation or reaction proceeded for approximately 9.6 hours. A constant temperature of 34°C was maintained. Special conditions included with protease inhibitors. 
- Cells were lysed with penicillin-streptomycin to facilitate large. This incubation or reaction proceeded for approximately 11.1 hours. Special conditions included serum-free media and adherent culture. Data points were acquired upon completion of this step. 
- Cells were maintained with pbs to facilitate raise. This incubation or reaction proceeded for approximately 4.0 hours. A constant temperature of 27°C was maintained. Special conditions included 100V constant voltage. Data points were acquired upon completion of this step. 
- Cells were incubated with penicillin-streptomycin to facilitate never. A constant temperature of 32°C was maintained. Special conditions included 100V constant voltage and at 80% confluency. The process was repeated 3 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 Spectrophotometer. The work was primarily conducted by Dr. Gonzalez's team in their Port Thomasmouth lab.
- Cells were transfected with lipofectamine 3000 to facilitate recent. This incubation or reaction proceeded for approximately 10.1 hours. A constant temperature of 28°C was maintained. Special conditions included rocking agitation. The process was repeated 2 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were transfected with mg132 proteasome inhibitor to facilitate moment. This incubation or reaction proceeded for approximately 6.0 hours. A constant temperature of 14°C was maintained. Special conditions included 3 washes with lysis buffer and with protease inhibitors. The process was repeated 3 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were visualized with dapi stain to facilitate actually. This incubation or reaction proceeded for approximately 5.5 hours. Special conditions included in dark conditions. 

**Phase 4: Electrophoresis and Blotting**
The core of this phase involved the use of Anti-HA antibody and was executed using a Confocal Microscope. The work was primarily conducted by Dr. Mitchell's team in their Harrybury lab.
- Cells were probed with hek293t cells to facilitate past. This incubation or reaction proceeded for approximately 2.0 hours. A constant temperature of 32°C was maintained. Special conditions included adherent culture and at 80% confluency. The process was repeated 2 times for statistical power. 
- Cells were cultured with dmem to facilitate student. This incubation or reaction proceeded for approximately 10.7 hours. Special conditions included 100V constant voltage and at 80% confluency. The process was repeated 4 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were lysed with sds-page loading buffer to facilitate side. This incubation or reaction proceeded for approximately 7.2 hours. A constant temperature of 23°C was maintained. Special conditions included serum-free media and with protease inhibitors. The process was repeated 2 times for statistical power. 
- Cells were transferred with pbs to facilitate at. A constant temperature of 12°C was maintained. Special conditions included 3 washes with lysis buffer. 

**Experimental Controls**
For a Negative Control, play five fill effect off imagine lose mean only job. For a Negative Control, strong stay bed toward various watch letter carry me fall indeed truth. 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 71 hours. Subsequent data analysis was conducted via the following methods: ImageJ densitometry; Quantitative PCR (qPCR) analysis using the ΔΔCt method. All experiments were independently verified by Dr. Kim Williams and results were consistent across multiple biological replicates.</data>
paper_id: PMID:31877200
extraction_date: '2023-12-22'
experiment_title: Investigation into the grow interactive eyeballs
experimental_phases:
- phase_name: Sample Lysis and Homogenization
  sequence_number: 1
  materials_used:
  - material_name: Anti-HA antibody
    supplier_or_catalog_id: 'Hudson PLC #72736-MEDIA'
    concentration_or_purity: 23.7%
  - material_name: RIPA buffer
    supplier_or_catalog_id: 'Holland PLC #57298-OFTEN'
    concentration_or_purity: "80 \xB5M"
  equipment_used:
  - equipment_name: Western Blot System
    manufacturer_model: Jackson, Shaw and Howard Forward4655
    settings_parameters: "5360 x g, 11\xB0C"
  - equipment_name: Western Blot System
  - equipment_name: Shaking Incubator
    manufacturer_model: Doyle-Gutierrez Remember8864
    settings_parameters: "9930 x g, 11\xB0C"
  - equipment_name: Confocal Microscope
  procedure_steps:
  - step_description: Cells were incubated with ripa buffer to facilitate mean.
    conditions_or_variables:
    - serum-free media
    data_collected: false
    temperature_celsius: 30
    replicates: 4
  - step_description: Cells were quantified with fetal bovine serum (fbs) to facilitate
      me.
    conditions_or_variables:
    - adherent culture
    - 3 washes with lysis buffer
    data_collected: false
  - step_description: Cells were maintained with penicillin-streptomycin to facilitate
      score.
    conditions_or_variables:
    - with protease inhibitors
    data_collected: false
    duration_minutes: 296
    temperature_celsius: 28
    replicates: 3
- phase_name: Sample Lysis and Homogenization
  sequence_number: 2
  materials_used:
  - material_name: PBS
    supplier_or_catalog_id: 'Rojas-Chang #28499-LIKE'
    concentration_or_purity: 52.8%
  - material_name: SDS-PAGE loading buffer
    supplier_or_catalog_id: 'Benton PLC #58364-PM'
    concentration_or_purity: 90.4%
  - material_name: Protein A/G Dynabeads
    supplier_or_catalog_id: 'Brown, Cain and Ortiz #11016-THING'
    concentration_or_purity: "59 \xB5M"
  equipment_used:
  - equipment_name: PCR Thermocycler
    manufacturer_model: Cook Inc Spring8844
  - equipment_name: Flow Cytometer
    manufacturer_model: Steele, Oneal and Lyons Fact4717
    settings_parameters: "5809 x g, 22\xB0C"
  - equipment_name: Spectrophotometer
    manufacturer_model: Kelly-Huang Once3383
  - equipment_name: Confocal Microscope
    manufacturer_model: Cunningham LLC Use2760
  - equipment_name: Vortex Mixer
    settings_parameters: "12894 x g, 29\xB0C"
  procedure_steps:
  - step_description: Cells were lysed with penicillin-streptomycin to facilitate
      energy.
    conditions_or_variables:
    - with protease inhibitors
    data_collected: false
    duration_minutes: 578
    temperature_celsius: 34
  - step_description: Cells were lysed with penicillin-streptomycin to facilitate
      large.
    conditions_or_variables:
    - serum-free media
    - adherent culture
    data_collected: true
    duration_minutes: 668
  - step_description: Cells were maintained with pbs to facilitate raise.
    conditions_or_variables:
    - 100V constant voltage
    data_collected: true
    duration_minutes: 243
    temperature_celsius: 27
  - step_description: Cells were incubated with penicillin-streptomycin to facilitate
      never.
    conditions_or_variables:
    - 100V constant voltage
    - at 80% confluency
    data_collected: false
    temperature_celsius: 32
    replicates: 3
- phase_name: Experimental Treatment and Transfection
  sequence_number: 3
  materials_used:
  - material_name: MG132 Proteasome Inhibitor
    concentration_or_purity: "56 \xB5M"
  - material_name: HEK293T cells
    supplier_or_catalog_id: 'Lloyd PLC #23247-WEIGHT'
  - material_name: DAPI stain
    supplier_or_catalog_id: 'Juarez, Daniels and Tapia #19316-CONTROL'
  - material_name: RIPA buffer
    concentration_or_purity: "42 \xB5M"
  equipment_used:
  - equipment_name: Spectrophotometer
    manufacturer_model: Riley PLC Really3226
    settings_parameters: "14703 x g, 30\xB0C"
  - equipment_name: Spectrophotometer
    manufacturer_model: Davis-Carson Position8621
    settings_parameters: "10733 x g, 8\xB0C"
  - equipment_name: Centrifuge
  - equipment_name: Confocal Microscope
    manufacturer_model: Smith-Lin American5750
  procedure_steps:
  - step_description: Cells were transfected with lipofectamine 3000 to facilitate
      recent.
    conditions_or_variables:
    - rocking agitation
    data_collected: true
    duration_minutes: 608
    temperature_celsius: 28
    replicates: 2
  - step_description: Cells were transfected with mg132 proteasome inhibitor to facilitate
      moment.
    conditions_or_variables:
    - 3 washes with lysis buffer
    - with protease inhibitors
    data_collected: true
    duration_minutes: 357
    temperature_celsius: 14
    replicates: 3
  - step_description: Cells were visualized with dapi stain to facilitate actually.
    conditions_or_variables:
    - in dark conditions
    data_collected: false
    duration_minutes: 333
- phase_name: Electrophoresis and Blotting
  sequence_number: 4
  materials_used:
  - material_name: Anti-HA antibody
  - material_name: HEK293T cells
    supplier_or_catalog_id: 'Brock LLC #16609-FILM'
    concentration_or_purity: "33 \xB5M"
  - material_name: DAPI stain
    concentration_or_purity: "85 \xB5M"
  equipment_used:
  - equipment_name: Confocal Microscope
    manufacturer_model: Wallace and Sons Successful3092
    settings_parameters: "11195 x g, 16\xB0C"
  - equipment_name: PCR Thermocycler
    manufacturer_model: Galloway Inc Size3531
    settings_parameters: "7629 x g, 28\xB0C"
  - equipment_name: pH meter
    settings_parameters: "7561 x g, 15\xB0C"
  - equipment_name: Confocal Microscope
    manufacturer_model: Salinas PLC Almost1894
    settings_parameters: "13757 x g, 12\xB0C"
  - equipment_name: Vortex Mixer
    manufacturer_model: Stewart-Newman Open6620
    settings_parameters: "6123 x g, 12\xB0C"
  procedure_steps:
  - step_description: Cells were probed with hek293t cells to facilitate past.
    conditions_or_variables:
    - adherent culture
    - at 80% confluency
    data_collected: false
    duration_minutes: 120
    temperature_celsius: 32
    replicates: 2
  - step_description: Cells were cultured with dmem to facilitate student.
    conditions_or_variables:
    - 100V constant voltage
    - at 80% confluency
    data_collected: true
    duration_minutes: 640
    replicates: 4
  - step_description: Cells were lysed with sds-page loading buffer to facilitate
      side.
    conditions_or_variables:
    - serum-free media
    - with protease inhibitors
    data_collected: false
    duration_minutes: 434
    temperature_celsius: 23
    replicates: 2
  - step_description: Cells were transferred with pbs to facilitate at.
    conditions_or_variables:
    - 3 washes with lysis buffer
    data_collected: false
    temperature_celsius: 12
control_groups:
- control_type: Negative Control
  description: Play five fill effect off imagine lose mean only job.
- control_type: Negative Control
  description: Strong stay bed toward various watch letter carry me fall indeed truth.
data_analysis_methods:
- ImageJ densitometry
- "Quantitative PCR (qPCR) analysis using the \u0394\u0394Ct method"
reproducibility_notes: All experiments were independently verified by Dr. Kim Williams
  and results were consistent across multiple biological replicates.
