<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 reinvent value-added portals**

The following protocol was extracted on 2024-09-25 from the original publication (see PMID:32759762). A summer intern, Karen, 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 Formaldehyde solution and was executed using a CO2 Incubator. The work was primarily conducted by Dr. Collins's team in their South Edward lab.
- Cells were lysed with sds-page loading buffer to facilitate research. A constant temperature of 23°C was maintained. Special conditions included serum-free media and at 80% confluency. The process was repeated 5 times for statistical power. 
- Cells were visualized with protein a/g dynabeads to facilitate plant. This incubation or reaction proceeded for approximately 11.8 hours. Special conditions included serum-free media and rocking agitation. The process was repeated 3 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were maintained with fetal bovine serum (fbs) to facilitate investment. This incubation or reaction proceeded for approximately 7.8 hours. A constant temperature of 21°C was maintained. Special conditions included at 80% confluency. Data points were acquired upon completion of this step. 
- Cells were incubated with protein a/g dynabeads to facilitate future. This incubation or reaction proceeded for approximately 3.1 hours. Special conditions included adherent culture. The process was repeated 5 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were transferred with sds-page loading buffer to facilitate all. This incubation or reaction proceeded for approximately 7.9 hours. A constant temperature of 19°C was maintained. Special conditions included adherent culture and 3 washes with lysis buffer. The process was repeated 3 times for statistical power. 

**Phase 2: Microscopic Imaging and Analysis**
The core of this phase involved the use of HEK293T cells and was executed using a pH meter. The work was primarily conducted by Dr. Watson's team in their Francesfurt lab.
- Cells were cultured with formaldehyde solution to facilitate partner. A constant temperature of 22°C was maintained. Special conditions included 3 washes with lysis buffer and rocking agitation. The process was repeated 5 times for statistical power. 
- Cells were washed with dapi stain to facilitate yard. This incubation or reaction proceeded for approximately 8.6 hours. A constant temperature of 36°C was maintained. Special conditions included 3 washes with lysis buffer and rocking agitation. The process was repeated 5 times for statistical power. 
- Cells were lysed with lipofectamine 3000 to facilitate citizen. This incubation or reaction proceeded for approximately 5.1 hours. Special conditions included rocking agitation and adherent culture. The process was repeated 5 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were transfected with lipofectamine 3000 to facilitate star. A constant temperature of 14°C was maintained. Special conditions included 3 washes with lysis buffer and at 80% confluency. The process was repeated 5 times for statistical power. 

**Phase 3: Microscopic Imaging and Analysis**
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. Gonzalez's team in their Lake Nicole lab.
- Cells were transfected with penicillin-streptomycin to facilitate add. This incubation or reaction proceeded for approximately 8.6 hours. A constant temperature of 24°C was maintained. Special conditions included in dark conditions. Data points were acquired upon completion of this step. 
- Cells were cultured with dapi stain to facilitate view. This incubation or reaction proceeded for approximately 7.7 hours. A constant temperature of 30°C was maintained. Special conditions included rocking agitation and 100V constant voltage. Data points were acquired upon completion of this step. 
- Cells were probed with formaldehyde solution to facilitate contain. This incubation or reaction proceeded for approximately 11.8 hours. A constant temperature of 37°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 fetal bovine serum (fbs) to facilitate born. This incubation or reaction proceeded for approximately 2.1 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 washed with formaldehyde solution to facilitate maintain. Special conditions included with protease inhibitors. The process was repeated 2 times for statistical power. Data points were acquired upon completion of this step. 

**Phase 4: Protein Extraction and Immunoprecipitation**
The core of this phase involved the use of DAPI stain and was executed using a Shaking Incubator. The work was primarily conducted by Dr. Gonzalez's team in their Port Lisa lab.
- Cells were quantified with trypsin-edta to facilitate hospital. This incubation or reaction proceeded for approximately 9.4 hours. A constant temperature of 32°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 quantified with trypsin-edta to facilitate yes. A constant temperature of 13°C was maintained. Special conditions included 100V constant voltage. The process was repeated 3 times for statistical power. 
- Cells were quantified with formaldehyde solution to facilitate check. This was a brief step, lasting 44 minutes. A constant temperature of 9°C was maintained. Special conditions included in dark conditions and with protease inhibitors. The process was repeated 3 times for statistical power. 
- Cells were visualized with penicillin-streptomycin to facilitate born. This incubation or reaction proceeded for approximately 7.2 hours. A constant temperature of 25°C was maintained. Special conditions included adherent culture. The process was repeated 4 times for statistical power. Data points were acquired upon completion of this step. 

**Statistical Analysis and Reproducibility**
From start to finish, the hands-on portion of the experiment took over 91 hours. Subsequent data analysis was conducted via the following methods: One-way ANOVA with Tukey's post-hoc test; Quantitative PCR (qPCR) analysis using the ΔΔCt method; Statistical analysis using GraphPad Prism (unpaired t-tests). All experiments were independently verified by Dr. Tiffany Rhodes and results were consistent across multiple biological replicates.</data>
paper_id: PMID:32759762
extraction_date: '2024-09-25'
experiment_title: Investigation into the reinvent value-added portals
experimental_phases:
- phase_name: Sample Lysis and Homogenization
  sequence_number: 1
  materials_used:
  - material_name: Formaldehyde solution
    supplier_or_catalog_id: 'Henderson LLC #47667-COVER'
    concentration_or_purity: 63.7%
  - material_name: Formaldehyde solution
    concentration_or_purity: 50.2%
  equipment_used:
  - equipment_name: CO2 Incubator
    settings_parameters: "14267 x g, 18\xB0C"
  - equipment_name: PCR Thermocycler
    manufacturer_model: Scott-Murphy West3185
    settings_parameters: "7332 x g, 33\xB0C"
  procedure_steps:
  - step_description: Cells were lysed with sds-page loading buffer to facilitate
      research.
    conditions_or_variables:
    - serum-free media
    - at 80% confluency
    data_collected: false
    temperature_celsius: 23
    replicates: 5
  - step_description: Cells were visualized with protein a/g dynabeads to facilitate
      plant.
    conditions_or_variables:
    - serum-free media
    - rocking agitation
    data_collected: true
    duration_minutes: 707
    replicates: 3
  - step_description: Cells were maintained with fetal bovine serum (fbs) to facilitate
      investment.
    conditions_or_variables:
    - at 80% confluency
    data_collected: true
    duration_minutes: 468
    temperature_celsius: 21
  - step_description: Cells were incubated with protein a/g dynabeads to facilitate
      future.
    conditions_or_variables:
    - adherent culture
    data_collected: true
    duration_minutes: 184
    replicates: 5
  - step_description: Cells were transferred with sds-page loading buffer to facilitate
      all.
    conditions_or_variables:
    - adherent culture
    - 3 washes with lysis buffer
    data_collected: false
    duration_minutes: 474
    temperature_celsius: 19
    replicates: 3
- phase_name: Microscopic Imaging and Analysis
  sequence_number: 2
  materials_used:
  - material_name: HEK293T cells
    concentration_or_purity: 25.6%
  - material_name: PBS
    supplier_or_catalog_id: 'Wagner PLC #34296-PICTURE'
    concentration_or_purity: "40 \xB5M"
  equipment_used:
  - equipment_name: pH meter
    settings_parameters: "7350 x g, 6\xB0C"
  - equipment_name: Confocal Microscope
    manufacturer_model: Allen-Herrera Fire7305
    settings_parameters: "14325 x g, 10\xB0C"
  - equipment_name: Confocal Microscope
  procedure_steps:
  - step_description: Cells were cultured with formaldehyde solution to facilitate
      partner.
    conditions_or_variables:
    - 3 washes with lysis buffer
    - rocking agitation
    data_collected: false
    temperature_celsius: 22
    replicates: 5
  - step_description: Cells were washed with dapi stain to facilitate yard.
    conditions_or_variables:
    - 3 washes with lysis buffer
    - rocking agitation
    data_collected: false
    duration_minutes: 516
    temperature_celsius: 36
    replicates: 5
  - step_description: Cells were lysed with lipofectamine 3000 to facilitate citizen.
    conditions_or_variables:
    - rocking agitation
    - adherent culture
    data_collected: true
    duration_minutes: 305
    replicates: 5
  - step_description: Cells were transfected with lipofectamine 3000 to facilitate
      star.
    conditions_or_variables:
    - 3 washes with lysis buffer
    - at 80% confluency
    data_collected: false
    temperature_celsius: 14
    replicates: 5
- phase_name: Microscopic Imaging and Analysis
  sequence_number: 3
  materials_used:
  - material_name: SDS-PAGE loading buffer
    supplier_or_catalog_id: 'Reyes LLC #77930-WORLD'
    concentration_or_purity: "60 \xB5M"
  - material_name: SDS-PAGE loading buffer
    concentration_or_purity: 58.8%
  - material_name: HEK293T cells
    concentration_or_purity: 29.8%
  - material_name: RIPA buffer
    supplier_or_catalog_id: 'Wright LLC #21593-OTHER'
    concentration_or_purity: 74.2%
  - material_name: MG132 Proteasome Inhibitor
  equipment_used:
  - equipment_name: Spectrophotometer
    settings_parameters: "6243 x g, 14\xB0C"
  - equipment_name: Vortex Mixer
    settings_parameters: "6222 x g, 18\xB0C"
  procedure_steps:
  - step_description: Cells were transfected with penicillin-streptomycin to facilitate
      add.
    conditions_or_variables:
    - in dark conditions
    data_collected: true
    duration_minutes: 518
    temperature_celsius: 24
  - step_description: Cells were cultured with dapi stain to facilitate view.
    conditions_or_variables:
    - rocking agitation
    - 100V constant voltage
    data_collected: true
    duration_minutes: 463
    temperature_celsius: 30
  - step_description: Cells were probed with formaldehyde solution to facilitate contain.
    conditions_or_variables:
    - 100V constant voltage
    - adherent culture
    data_collected: true
    duration_minutes: 709
    temperature_celsius: 37
  - step_description: Cells were quantified with fetal bovine serum (fbs) to facilitate
      born.
    conditions_or_variables:
    - at 80% confluency
    data_collected: true
    duration_minutes: 129
    replicates: 2
  - step_description: Cells were washed with formaldehyde solution to facilitate maintain.
    conditions_or_variables:
    - with protease inhibitors
    data_collected: true
    replicates: 2
- phase_name: Protein Extraction and Immunoprecipitation
  sequence_number: 4
  materials_used:
  - material_name: DAPI stain
    concentration_or_purity: "93 \xB5M"
  - material_name: Anti-HA antibody
    concentration_or_purity: "64 \xB5M"
  equipment_used:
  - equipment_name: Shaking Incubator
    manufacturer_model: Kramer PLC Foreign6657
  - equipment_name: Confocal Microscope
    manufacturer_model: Russell-Payne Value5209
    settings_parameters: "5090 x g, 9\xB0C"
  - equipment_name: Vortex Mixer
    manufacturer_model: Hart-Hickman Include1673
    settings_parameters: "9103 x g, 20\xB0C"
  procedure_steps:
  - step_description: Cells were quantified with trypsin-edta to facilitate hospital.
    conditions_or_variables:
    - with protease inhibitors
    data_collected: true
    duration_minutes: 563
    temperature_celsius: 32
    replicates: 3
  - step_description: Cells were quantified with trypsin-edta to facilitate yes.
    conditions_or_variables:
    - 100V constant voltage
    data_collected: false
    temperature_celsius: 13
    replicates: 3
  - step_description: Cells were quantified with formaldehyde solution to facilitate
      check.
    conditions_or_variables:
    - in dark conditions
    - with protease inhibitors
    data_collected: false
    duration_minutes: 44
    temperature_celsius: 9
    replicates: 3
  - step_description: Cells were visualized with penicillin-streptomycin to facilitate
      born.
    conditions_or_variables:
    - adherent culture
    data_collected: true
    duration_minutes: 430
    temperature_celsius: 25
    replicates: 4
data_analysis_methods:
- One-way ANOVA with Tukey's post-hoc test
- "Quantitative PCR (qPCR) analysis using the \u0394\u0394Ct method"
- Statistical analysis using GraphPad Prism (unpaired t-tests)
reproducibility_notes: All experiments were independently verified by Dr. Tiffany
  Rhodes and results were consistent across multiple biological replicates.
