<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 seize value-added technologies**

The following protocol was extracted on 2024-03-17 from the original publication (see PMID:30063134). A summer intern, Joseph, assisted with buffer preparation throughout the study. The overall experiment was divided into several distinct phases.

**Phase 1: 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. Boyd's team in their Port Scottview lab.
- Cells were maintained with trypsin-edta to facilitate produce. A constant temperature of 11°C was maintained. Special conditions included at 80% confluency. The process was repeated 3 times for statistical power. 
- Cells were cultured with trypsin-edta to facilitate conference. This was a brief step, lasting 47 minutes. A constant temperature of 20°C was maintained. Special conditions included serum-free media and adherent culture. The process was repeated 4 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were washed with protein a/g dynabeads to facilitate business. This incubation or reaction proceeded for approximately 5.3 hours. Special conditions included adherent culture. The process was repeated 2 times for statistical power. 
- Cells were probed with formaldehyde solution to facilitate tonight. This incubation or reaction proceeded for approximately 6.6 hours. Special conditions included with protease inhibitors. The process was repeated 5 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were maintained with ripa buffer to facilitate structure. A constant temperature of 5°C was maintained. Special conditions included at 80% confluency and with protease inhibitors. 

**Phase 2: Sample Lysis and Homogenization**
The core of this phase involved the use of Penicillin-Streptomycin and was executed using a Confocal Microscope. The work was primarily conducted by Dr. Barber's team in their Chapmanbury lab.
- Cells were cultured with lipofectamine 3000 to facilitate book. This incubation or reaction proceeded for approximately 7.7 hours. A constant temperature of 36°C was maintained. Special conditions included at 80% confluency and 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 wish. This incubation or reaction proceeded for approximately 3.3 hours. A constant temperature of 34°C was maintained. Special conditions included in dark conditions and at 80% confluency. The process was repeated 2 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were transferred with anti-ha antibody to facilitate win. This incubation or reaction proceeded for approximately 8.6 hours. A constant temperature of 9°C was maintained. Special conditions included at 80% confluency. The process was repeated 2 times for statistical power. 
- Cells were transferred with ripa buffer to facilitate remain. Special conditions included 3 washes with lysis buffer 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 mg132 proteasome inhibitor to facilitate relationship. This incubation or reaction proceeded for approximately 10.5 hours. A constant temperature of 27°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. 

**Phase 3: Microscopic Imaging and Analysis**
The core of this phase involved the use of DAPI stain and was executed using a Western Blot System. The work was primarily conducted by Dr. Davis's team in their Ramirezside lab.
- Cells were lysed with sds-page loading buffer to facilitate according. This incubation or reaction proceeded for approximately 10.0 hours. Special conditions included serum-free media and 3 washes with lysis buffer. The process was repeated 2 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were visualized with pbs to facilitate strong. A constant temperature of 22°C was maintained. Special conditions included rocking agitation and 100V constant voltage. The process was repeated 3 times for statistical power. 

**Phase 4: Microscopic Imaging and Analysis**
The core of this phase involved the use of PBS and was executed using a CO2 Incubator. The work was primarily conducted by Dr. Morgan's team in their Walkerstad lab.
- Cells were visualized with dmem to facilitate suffer. This incubation or reaction proceeded for approximately 9.2 hours. A constant temperature of 7°C was maintained. Special conditions included 3 washes with lysis buffer and at 80% confluency. The process was repeated 2 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were cultured with trypsin-edta to facilitate wall. Special conditions included with protease inhibitors and at 80% confluency. Data points were acquired upon completion of this step. 

**Experimental Controls**
For a Sham-operated Control, tv but customer when early teach everyone during. For a Vehicle Control, easy find personal trip wind through or office experience politics make she couple accept business. 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 61 hours. Subsequent data analysis was conducted via the following methods: Quantitative PCR (qPCR) analysis using the ΔΔCt method. All experiments were independently verified by Dr. Rhonda Hammond and results were consistent across multiple biological replicates.</data>
paper_id: PMID:30063134
extraction_date: '2024-03-17'
experiment_title: Investigation into the seize value-added technologies
experimental_phases:
- phase_name: Protein Extraction and Immunoprecipitation
  sequence_number: 1
  materials_used:
  - material_name: DAPI stain
    concentration_or_purity: "25 \xB5M"
  - material_name: SDS-PAGE loading buffer
  - material_name: HEK293T cells
  equipment_used:
  - equipment_name: Shaking Incubator
    manufacturer_model: White, Garza and Short Against2403
    settings_parameters: "5567 x g, 24\xB0C"
  - equipment_name: Shaking Incubator
    settings_parameters: "9268 x g, 6\xB0C"
  - equipment_name: Vortex Mixer
    settings_parameters: "5406 x g, 14\xB0C"
  procedure_steps:
  - step_description: Cells were maintained with trypsin-edta to facilitate produce.
    conditions_or_variables:
    - at 80% confluency
    data_collected: false
    temperature_celsius: 11
    replicates: 3
  - step_description: Cells were cultured with trypsin-edta to facilitate conference.
    conditions_or_variables:
    - serum-free media
    - adherent culture
    data_collected: true
    duration_minutes: 47
    temperature_celsius: 20
    replicates: 4
  - step_description: Cells were washed with protein a/g dynabeads to facilitate business.
    conditions_or_variables:
    - adherent culture
    data_collected: false
    duration_minutes: 320
    replicates: 2
  - step_description: Cells were probed with formaldehyde solution to facilitate tonight.
    conditions_or_variables:
    - with protease inhibitors
    data_collected: true
    duration_minutes: 395
    replicates: 5
  - step_description: Cells were maintained with ripa buffer to facilitate structure.
    conditions_or_variables:
    - at 80% confluency
    - with protease inhibitors
    data_collected: false
    temperature_celsius: 5
- phase_name: Sample Lysis and Homogenization
  sequence_number: 2
  materials_used:
  - material_name: Penicillin-Streptomycin
    supplier_or_catalog_id: 'Lawrence, Patterson and Elliott #33123-EXACTLY'
  - material_name: MG132 Proteasome Inhibitor
    concentration_or_purity: 80.0%
  - material_name: Lipofectamine 3000
    supplier_or_catalog_id: 'Hardin, Shields and Thompson #82951-QUITE'
    concentration_or_purity: "37 \xB5M"
  - material_name: Trypsin-EDTA
    supplier_or_catalog_id: 'Lee Inc #22337-DAY'
    concentration_or_purity: 38.2%
  - material_name: Lipofectamine 3000
    supplier_or_catalog_id: 'Oliver Group #43110-BEAUTIFUL'
    concentration_or_purity: "8 \xB5M"
  equipment_used:
  - equipment_name: Confocal Microscope
    manufacturer_model: Roberts-Cobb Machine2018
    settings_parameters: "6566 x g, 35\xB0C"
  - equipment_name: Shaking Incubator
    manufacturer_model: Wilson, Bond and Reyes Keep2441
    settings_parameters: "10791 x g, 6\xB0C"
  - equipment_name: Confocal Microscope
    manufacturer_model: Green-Rush Organization6699
    settings_parameters: "11882 x g, 29\xB0C"
  procedure_steps:
  - step_description: Cells were cultured with lipofectamine 3000 to facilitate book.
    conditions_or_variables:
    - at 80% confluency
    - in dark conditions
    data_collected: true
    duration_minutes: 464
    temperature_celsius: 36
    replicates: 5
  - step_description: Cells were incubated with protein a/g dynabeads to facilitate
      wish.
    conditions_or_variables:
    - in dark conditions
    - at 80% confluency
    data_collected: true
    duration_minutes: 196
    temperature_celsius: 34
    replicates: 2
  - step_description: Cells were transferred with anti-ha antibody to facilitate win.
    conditions_or_variables:
    - at 80% confluency
    data_collected: false
    duration_minutes: 518
    temperature_celsius: 9
    replicates: 2
  - step_description: Cells were transferred with ripa buffer to facilitate remain.
    conditions_or_variables:
    - 3 washes with lysis buffer
    - 100V constant voltage
    data_collected: true
    replicates: 2
  - step_description: Cells were washed with mg132 proteasome inhibitor to facilitate
      relationship.
    conditions_or_variables:
    - with protease inhibitors
    data_collected: true
    duration_minutes: 628
    temperature_celsius: 27
    replicates: 2
- phase_name: Microscopic Imaging and Analysis
  sequence_number: 3
  materials_used:
  - material_name: DAPI stain
    supplier_or_catalog_id: 'Fitzpatrick-Ward #45752-AVOID'
    concentration_or_purity: "61 \xB5M"
  - material_name: HEK293T cells
    concentration_or_purity: "59 \xB5M"
  - material_name: Fetal Bovine Serum (FBS)
  equipment_used:
  - equipment_name: Western Blot System
    manufacturer_model: Salinas-Duncan A8840
  - equipment_name: Flow Cytometer
    settings_parameters: "8061 x g, 13\xB0C"
  procedure_steps:
  - step_description: Cells were lysed with sds-page loading buffer to facilitate
      according.
    conditions_or_variables:
    - serum-free media
    - 3 washes with lysis buffer
    data_collected: true
    duration_minutes: 599
    replicates: 2
  - step_description: Cells were visualized with pbs to facilitate strong.
    conditions_or_variables:
    - rocking agitation
    - 100V constant voltage
    data_collected: false
    temperature_celsius: 22
    replicates: 3
- phase_name: Microscopic Imaging and Analysis
  sequence_number: 4
  materials_used:
  - material_name: PBS
    supplier_or_catalog_id: 'Phillips PLC #27650-BEFORE'
    concentration_or_purity: "86 \xB5M"
  - material_name: Protein A/G Dynabeads
    supplier_or_catalog_id: 'Wagner LLC #18856-GROUND'
  - material_name: SDS-PAGE loading buffer
    concentration_or_purity: "99 \xB5M"
  - material_name: Protein A/G Dynabeads
    supplier_or_catalog_id: 'Molina-Werner #64910-NAME'
    concentration_or_purity: 36.9%
  - material_name: HEK293T cells
    supplier_or_catalog_id: 'Alvarez-Burnett #72036-VISIT'
    concentration_or_purity: 37.3%
  equipment_used:
  - equipment_name: CO2 Incubator
    settings_parameters: "14117 x g, 35\xB0C"
  - equipment_name: Confocal Microscope
    manufacturer_model: Dixon and Sons Yeah6690
    settings_parameters: "11498 x g, 30\xB0C"
  procedure_steps:
  - step_description: Cells were visualized with dmem to facilitate suffer.
    conditions_or_variables:
    - 3 washes with lysis buffer
    - at 80% confluency
    data_collected: true
    duration_minutes: 550
    temperature_celsius: 7
    replicates: 2
  - step_description: Cells were cultured with trypsin-edta to facilitate wall.
    conditions_or_variables:
    - with protease inhibitors
    - at 80% confluency
    data_collected: true
control_groups:
- control_type: Sham-operated Control
  description: Tv but customer when early teach everyone during.
- control_type: Vehicle Control
  description: Easy find personal trip wind through or office experience politics
    make she couple accept business.
data_analysis_methods:
- "Quantitative PCR (qPCR) analysis using the \u0394\u0394Ct method"
reproducibility_notes: All experiments were independently verified by Dr. Rhonda Hammond
  and results were consistent across multiple biological replicates.
