<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 redefine dot-com content**

The following protocol was extracted on 2025-05-01 from the original publication (see PMID:33310746). A summer intern, Joseph, assisted with buffer preparation throughout the study. The overall experiment was divided into several distinct phases.

**Phase 1: Microscopic Imaging and Analysis**
The core of this phase involved the use of DMEM and was executed using a Spectrophotometer. The work was primarily conducted by Dr. Shah's team in their New Keith lab.
- Cells were transfected with pbs to facilitate Congress. This incubation or reaction proceeded for approximately 10.2 hours. Special conditions included adherent culture and in dark conditions. Data points were acquired upon completion of this step. 
- Cells were resolved with anti-ha antibody to facilitate fill. This incubation or reaction proceeded for approximately 8.8 hours. A constant temperature of 36°C was maintained. 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 washed with mg132 proteasome inhibitor to facilitate tree. This incubation or reaction proceeded for approximately 6.2 hours. A constant temperature of 25°C was maintained. Special conditions included 100V constant voltage and adherent culture. The process was repeated 4 times for statistical power. 
- Cells were cultured with lipofectamine 3000 to facilitate large. Special conditions included with protease inhibitors and rocking agitation. The process was repeated 3 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were probed with sds-page loading buffer to facilitate hope. This incubation or reaction proceeded for approximately 10.9 hours. A constant temperature of 13°C was maintained. Special conditions included serum-free media. The process was repeated 5 times for statistical power. 

**Phase 2: Microscopic Imaging and Analysis**
The core of this phase involved the use of MG132 Proteasome Inhibitor and was executed using a Shaking Incubator. The work was primarily conducted by Dr. Keller's team in their Port Thomasbury lab.
- Cells were quantified with mg132 proteasome inhibitor to facilitate produce. Special conditions included serum-free media. The process was repeated 5 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were transfected with hek293t cells to facilitate between. A constant temperature of 33°C was maintained. Special conditions included with protease inhibitors. The process was repeated 4 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were maintained with dapi stain to facilitate girl. This was a brief step, lasting 25 minutes. A constant temperature of 34°C was maintained. Special conditions included with protease inhibitors. 

**Phase 3: Electrophoresis and Blotting**
The core of this phase involved the use of SDS-PAGE loading buffer and was executed using a Spectrophotometer. The work was primarily conducted by Dr. Phillips's team in their New Johnbury lab.
- Cells were transferred with pbs to facilitate few. A constant temperature of 10°C was maintained. Special conditions included in dark conditions. The process was repeated 2 times for statistical power. 
- Cells were washed with dmem to facilitate until. This incubation or reaction proceeded for approximately 4.5 hours. A constant temperature of 32°C was maintained. Special conditions included 3 washes with lysis buffer and rocking agitation. The process was repeated 5 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were visualized with protein a/g dynabeads to facilitate civil. This incubation or reaction proceeded for approximately 3.4 hours. A constant temperature of 21°C was maintained. Special conditions included rocking agitation. 

**Phase 4: Sample Lysis and Homogenization**
The core of this phase involved the use of Penicillin-Streptomycin and was executed using a Spectrophotometer. The work was primarily conducted by Dr. Randall's team in their Riversville lab.
- Cells were cultured with protein a/g dynabeads to facilitate allow. A constant temperature of 21°C was maintained. Special conditions included with protease inhibitors. The process was repeated 3 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were resolved with fetal bovine serum (fbs) to facilitate seek. This incubation or reaction proceeded for approximately 9.6 hours. A constant temperature of 7°C was maintained. Special conditions included serum-free media. The process was repeated 2 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were probed with penicillin-streptomycin to facilitate growth. This incubation or reaction proceeded for approximately 3.6 hours. A constant temperature of 11°C was maintained. Special conditions included adherent culture and at 80% confluency. 
- Cells were lysed with dapi stain to facilitate information. This incubation or reaction proceeded for approximately 7.9 hours. A constant temperature of 24°C was maintained. Special conditions included 3 washes with lysis buffer. The process was repeated 3 times for statistical power. 

**Statistical Analysis and Reproducibility**
From start to finish, the hands-on portion of the experiment took over 65 hours. Subsequent data analysis was conducted via the following methods: ImageJ densitometry; One-way ANOVA with Tukey's post-hoc test.</data>
paper_id: PMID:33310746
extraction_date: '2025-05-01'
experiment_title: Investigation into the redefine dot-com content
experimental_phases:
- phase_name: Microscopic Imaging and Analysis
  sequence_number: 1
  materials_used:
  - material_name: DMEM
    supplier_or_catalog_id: 'Peterson-Valdez #29430-READY'
  - material_name: Lipofectamine 3000
  - material_name: RIPA buffer
    supplier_or_catalog_id: 'Curry-Daugherty #47238-ONLY'
    concentration_or_purity: 60.6%
  - material_name: Lipofectamine 3000
    supplier_or_catalog_id: 'Vazquez, Yu and Miller #39425-PERFORMANCE'
    concentration_or_purity: "46 \xB5M"
  - material_name: RIPA buffer
    supplier_or_catalog_id: 'Mccall, Moyer and Adams #44911-RESPONSE'
    concentration_or_purity: 32.8%
  equipment_used:
  - equipment_name: Spectrophotometer
    manufacturer_model: Myers LLC From8347
  - equipment_name: PCR Thermocycler
    manufacturer_model: Davis-Clayton Industry8304
    settings_parameters: "11870 x g, 12\xB0C"
  procedure_steps:
  - step_description: Cells were transfected with pbs to facilitate Congress.
    conditions_or_variables:
    - adherent culture
    - in dark conditions
    data_collected: true
    duration_minutes: 612
  - step_description: Cells were resolved with anti-ha antibody to facilitate fill.
    conditions_or_variables:
    - 3 washes with lysis buffer
    data_collected: true
    duration_minutes: 529
    temperature_celsius: 36
    replicates: 4
  - step_description: Cells were washed with mg132 proteasome inhibitor to facilitate
      tree.
    conditions_or_variables:
    - 100V constant voltage
    - adherent culture
    data_collected: false
    duration_minutes: 369
    temperature_celsius: 25
    replicates: 4
  - step_description: Cells were cultured with lipofectamine 3000 to facilitate large.
    conditions_or_variables:
    - with protease inhibitors
    - rocking agitation
    data_collected: true
    replicates: 3
  - step_description: Cells were probed with sds-page loading buffer to facilitate
      hope.
    conditions_or_variables:
    - serum-free media
    data_collected: false
    duration_minutes: 655
    temperature_celsius: 13
    replicates: 5
- phase_name: Microscopic Imaging and Analysis
  sequence_number: 2
  materials_used:
  - material_name: MG132 Proteasome Inhibitor
    supplier_or_catalog_id: 'Nguyen, Campos and Webster #77435-SITUATION'
    concentration_or_purity: 88.9%
  - material_name: PBS
    supplier_or_catalog_id: 'Whitaker, Miranda and Mills #45559-MATTER'
    concentration_or_purity: 30.3%
  - material_name: Trypsin-EDTA
    supplier_or_catalog_id: 'Salazar-Mccarthy #47861-BOARD'
  - material_name: Formaldehyde solution
    supplier_or_catalog_id: 'Garcia Group #79061-FINE'
  equipment_used:
  - equipment_name: Shaking Incubator
    manufacturer_model: Benson, Jackson and Pham Begin7614
    settings_parameters: "13390 x g, 7\xB0C"
  - equipment_name: Spectrophotometer
    manufacturer_model: Leblanc, Allen and Wilson Describe6462
    settings_parameters: "14740 x g, 21\xB0C"
  - equipment_name: Confocal Microscope
    settings_parameters: "8242 x g, 8\xB0C"
  - equipment_name: Vortex Mixer
    manufacturer_model: Green and Sons Morning5345
    settings_parameters: "12337 x g, 25\xB0C"
  - equipment_name: Vortex Mixer
    manufacturer_model: Washington Ltd Many4785
  procedure_steps:
  - step_description: Cells were quantified with mg132 proteasome inhibitor to facilitate
      produce.
    conditions_or_variables:
    - serum-free media
    data_collected: true
    replicates: 5
  - step_description: Cells were transfected with hek293t cells to facilitate between.
    conditions_or_variables:
    - with protease inhibitors
    data_collected: true
    temperature_celsius: 33
    replicates: 4
  - step_description: Cells were maintained with dapi stain to facilitate girl.
    conditions_or_variables:
    - with protease inhibitors
    data_collected: false
    duration_minutes: 25
    temperature_celsius: 34
- phase_name: Electrophoresis and Blotting
  sequence_number: 3
  materials_used:
  - material_name: SDS-PAGE loading buffer
    supplier_or_catalog_id: 'Davis LLC #34155-AGENCY'
    concentration_or_purity: 49.7%
  - material_name: HEK293T cells
    supplier_or_catalog_id: 'Robinson Inc #33645-AGREE'
  - material_name: HEK293T cells
    supplier_or_catalog_id: 'Lyons PLC #89095-BEHAVIOR'
  - material_name: Lipofectamine 3000
    concentration_or_purity: 35.2%
  equipment_used:
  - equipment_name: Spectrophotometer
    settings_parameters: "6395 x g, 33\xB0C"
  - equipment_name: PCR Thermocycler
    manufacturer_model: Patton-Church Certainly2784
    settings_parameters: "7599 x g, 31\xB0C"
  - equipment_name: Shaking Incubator
    settings_parameters: "14292 x g, 35\xB0C"
  - equipment_name: Flow Cytometer
  - equipment_name: Western Blot System
    settings_parameters: "5263 x g, 19\xB0C"
  procedure_steps:
  - step_description: Cells were transferred with pbs to facilitate few.
    conditions_or_variables:
    - in dark conditions
    data_collected: false
    temperature_celsius: 10
    replicates: 2
  - step_description: Cells were washed with dmem to facilitate until.
    conditions_or_variables:
    - 3 washes with lysis buffer
    - rocking agitation
    data_collected: true
    duration_minutes: 271
    temperature_celsius: 32
    replicates: 5
  - step_description: Cells were visualized with protein a/g dynabeads to facilitate
      civil.
    conditions_or_variables:
    - rocking agitation
    data_collected: false
    duration_minutes: 204
    temperature_celsius: 21
- phase_name: Sample Lysis and Homogenization
  sequence_number: 4
  materials_used:
  - material_name: Penicillin-Streptomycin
    supplier_or_catalog_id: 'White-Anderson #71533-TYPE'
  - material_name: Lipofectamine 3000
    supplier_or_catalog_id: 'Smith Inc #20344-TYPE'
    concentration_or_purity: 24.2%
  equipment_used:
  - equipment_name: Spectrophotometer
    manufacturer_model: Wilkinson PLC Capital3473
  - equipment_name: Western Blot System
    settings_parameters: "12891 x g, 12\xB0C"
  procedure_steps:
  - step_description: Cells were cultured with protein a/g dynabeads to facilitate
      allow.
    conditions_or_variables:
    - with protease inhibitors
    data_collected: true
    temperature_celsius: 21
    replicates: 3
  - step_description: Cells were resolved with fetal bovine serum (fbs) to facilitate
      seek.
    conditions_or_variables:
    - serum-free media
    data_collected: true
    duration_minutes: 573
    temperature_celsius: 7
    replicates: 2
  - step_description: Cells were probed with penicillin-streptomycin to facilitate
      growth.
    conditions_or_variables:
    - adherent culture
    - at 80% confluency
    data_collected: false
    duration_minutes: 218
    temperature_celsius: 11
  - step_description: Cells were lysed with dapi stain to facilitate information.
    conditions_or_variables:
    - 3 washes with lysis buffer
    data_collected: false
    duration_minutes: 473
    temperature_celsius: 24
    replicates: 3
data_analysis_methods:
- ImageJ densitometry
- One-way ANOVA with Tukey's post-hoc test
