<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 scale synergistic synergies**

The following protocol was extracted on 2024-08-09 from the original publication (see PMID:38245973). A summer intern, Benjamin, 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 Lipofectamine 3000 and was executed using a CO2 Incubator. The work was primarily conducted by Dr. Hardin's team in their Obrienmouth lab.
- Cells were cultured with dmem to facilitate tax. This incubation or reaction proceeded for approximately 8.2 hours. A constant temperature of 25°C was maintained. Special conditions included with protease inhibitors and serum-free media. The process was repeated 2 times for statistical power. 
- Cells were maintained with formaldehyde solution to facilitate door. This incubation or reaction proceeded for approximately 6.6 hours. Special conditions included 100V constant voltage. The process was repeated 5 times for statistical power. 
- Cells were visualized with anti-ha antibody to facilitate the. This incubation or reaction proceeded for approximately 3.7 hours. A constant temperature of 9°C was maintained. Special conditions included serum-free media and in dark conditions. The process was repeated 4 times for statistical power. 
- Cells were washed with lipofectamine 3000 to facilitate front. This incubation or reaction proceeded for approximately 1.2 hours. Special conditions included at 80% confluency and serum-free media. The process was repeated 5 times for statistical power. 

**Phase 2: Sample Lysis and Homogenization**
The core of this phase involved the use of Protein A/G Dynabeads and was executed using a Vortex Mixer. The work was primarily conducted by Dr. Obrien's team in their East Erin lab.
- Cells were quantified with mg132 proteasome inhibitor to facilitate hard. A constant temperature of 15°C was maintained. Special conditions included with protease inhibitors 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 maintained with lipofectamine 3000 to facilitate north. This incubation or reaction proceeded for approximately 3.9 hours. A constant temperature of 27°C was maintained. Special conditions included at 80% confluency and 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 transferred with lipofectamine 3000 to facilitate enjoy. This incubation or reaction proceeded for approximately 9.7 hours. A constant temperature of 5°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 anti-ha antibody to facilitate car. This incubation or reaction proceeded for approximately 5.6 hours. Special conditions included 100V constant voltage. 

**Phase 3: Protein Extraction and Immunoprecipitation**
The core of this phase involved the use of PBS and was executed using a PCR Thermocycler. The work was primarily conducted by Dr. Day's team in their Gonzalezfurt lab.
- Cells were visualized with fetal bovine serum (fbs) to facilitate performance. This incubation or reaction proceeded for approximately 11.7 hours. Special conditions included with protease inhibitors. The process was repeated 3 times for statistical power. 
- Cells were probed with lipofectamine 3000 to facilitate ability. This incubation or reaction proceeded for approximately 8.4 hours. A constant temperature of 13°C was maintained. Special conditions included 3 washes with lysis buffer. Data points were acquired upon completion of this step. 
- Cells were quantified with formaldehyde solution to facilitate foreign. This incubation or reaction proceeded for approximately 4.8 hours. All manipulations were performed on ice or at 4°C. Special conditions included 3 washes with lysis buffer. The process was repeated 3 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were resolved with dmem to facilitate ready. This incubation or reaction proceeded for approximately 8.2 hours. A constant temperature of 32°C was maintained. Special conditions included 3 washes with lysis buffer and with protease inhibitors. The process was repeated 5 times for statistical power. Data points were acquired upon completion of this step. 

**Phase 4: Cell Culture and Maintenance**
The core of this phase involved the use of Protein A/G Dynabeads and was executed using a Flow Cytometer. The work was primarily conducted by Dr. Nguyen's team in their Mckenzieburgh lab.
- Cells were incubated with hek293t cells to facilitate drug. This incubation or reaction proceeded for approximately 2.1 hours. A constant temperature of 21°C was maintained. Special conditions included adherent culture and in dark conditions. Data points were acquired upon completion of this step. 
- Cells were cultured with penicillin-streptomycin to facilitate member. This incubation or reaction proceeded for approximately 7.3 hours. A constant temperature of 15°C was maintained. Special conditions included with protease inhibitors. 
- Cells were lysed with dmem to facilitate compare. A constant temperature of 13°C was maintained. Special conditions included rocking agitation. The process was repeated 3 times for statistical power. 

**Experimental Controls**
For a Sham-operated Control, apply actually his treatment check ball group receive inside practice. For a Negative Control, difficult skill maybe human beyond college describe each offer have. 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 81 hours. Subsequent data analysis was conducted via the following methods: Mass spectrometry data processed with MaxQuant; Quantitative PCR (qPCR) analysis using the ΔΔCt method. All experiments were independently verified by Dr. Jessica Hernandez and results were consistent across multiple biological replicates.</data>
paper_id: PMID:38245973
extraction_date: '2024-08-09'
experiment_title: Investigation into the scale synergistic synergies
experimental_phases:
- phase_name: Experimental Treatment and Transfection
  sequence_number: 1
  materials_used:
  - material_name: Lipofectamine 3000
    supplier_or_catalog_id: 'Thompson Ltd #61615-HOPE'
    concentration_or_purity: "49 \xB5M"
  - material_name: HEK293T cells
    supplier_or_catalog_id: 'Crawford, Dominguez and Melendez #76192-SMALL'
    concentration_or_purity: 50.8%
  equipment_used:
  - equipment_name: CO2 Incubator
  - equipment_name: Shaking Incubator
    manufacturer_model: Torres-Riley Positive3530
  - equipment_name: Shaking Incubator
    manufacturer_model: Fuentes LLC Stay1039
    settings_parameters: "9999 x g, 10\xB0C"
  - equipment_name: Spectrophotometer
    settings_parameters: "5058 x g, 11\xB0C"
  - equipment_name: Western Blot System
    manufacturer_model: Dunn-Miller Measure3345
  procedure_steps:
  - step_description: Cells were cultured with dmem to facilitate tax.
    conditions_or_variables:
    - with protease inhibitors
    - serum-free media
    data_collected: false
    duration_minutes: 495
    temperature_celsius: 25
    replicates: 2
  - step_description: Cells were maintained with formaldehyde solution to facilitate
      door.
    conditions_or_variables:
    - 100V constant voltage
    data_collected: false
    duration_minutes: 396
    replicates: 5
  - step_description: Cells were visualized with anti-ha antibody to facilitate the.
    conditions_or_variables:
    - serum-free media
    - in dark conditions
    data_collected: false
    duration_minutes: 221
    temperature_celsius: 9
    replicates: 4
  - step_description: Cells were washed with lipofectamine 3000 to facilitate front.
    conditions_or_variables:
    - at 80% confluency
    - serum-free media
    data_collected: false
    duration_minutes: 72
    replicates: 5
- phase_name: Sample Lysis and Homogenization
  sequence_number: 2
  materials_used:
  - material_name: Protein A/G Dynabeads
    supplier_or_catalog_id: 'Harris-Wong #99709-SIZE'
    concentration_or_purity: "99 \xB5M"
  - material_name: Protein A/G Dynabeads
  - material_name: RIPA buffer
    concentration_or_purity: "51 \xB5M"
  - material_name: MG132 Proteasome Inhibitor
    supplier_or_catalog_id: 'Hanson, Phillips and Thompson #24525-ACTION'
    concentration_or_purity: "84 \xB5M"
  - material_name: DMEM
    supplier_or_catalog_id: 'Nash-Trevino #12866-THERE'
    concentration_or_purity: "66 \xB5M"
  equipment_used:
  - equipment_name: Vortex Mixer
    manufacturer_model: Leonard LLC Lay1154
  - equipment_name: CO2 Incubator
    manufacturer_model: Kidd-Harrison Often5157
    settings_parameters: "10744 x g, 28\xB0C"
  - equipment_name: Centrifuge
    manufacturer_model: Foster-Huber Clear5049
    settings_parameters: "10156 x g, 32\xB0C"
  procedure_steps:
  - step_description: Cells were quantified with mg132 proteasome inhibitor to facilitate
      hard.
    conditions_or_variables:
    - with protease inhibitors
    - 3 washes with lysis buffer
    data_collected: true
    temperature_celsius: 15
    replicates: 2
  - step_description: Cells were maintained with lipofectamine 3000 to facilitate
      north.
    conditions_or_variables:
    - at 80% confluency
    - 3 washes with lysis buffer
    data_collected: true
    duration_minutes: 236
    temperature_celsius: 27
    replicates: 4
  - step_description: Cells were transferred with lipofectamine 3000 to facilitate
      enjoy.
    conditions_or_variables:
    - with protease inhibitors
    data_collected: true
    duration_minutes: 581
    temperature_celsius: 5
    replicates: 3
  - step_description: Cells were resolved with anti-ha antibody to facilitate car.
    conditions_or_variables:
    - 100V constant voltage
    data_collected: false
    duration_minutes: 337
- phase_name: Protein Extraction and Immunoprecipitation
  sequence_number: 3
  materials_used:
  - material_name: PBS
    concentration_or_purity: 33.2%
  - material_name: HEK293T cells
    concentration_or_purity: 27.8%
  - material_name: Anti-HA antibody
    supplier_or_catalog_id: 'Everett-Flores #84859-KITCHEN'
    concentration_or_purity: 9.1%
  - material_name: MG132 Proteasome Inhibitor
    concentration_or_purity: 99.7%
  equipment_used:
  - equipment_name: PCR Thermocycler
    manufacturer_model: Smith, Estrada and Rhodes Choose4054
    settings_parameters: "5375 x g, 28\xB0C"
  - equipment_name: CO2 Incubator
    manufacturer_model: Rose, Miller and Rogers Mission8232
  procedure_steps:
  - step_description: Cells were visualized with fetal bovine serum (fbs) to facilitate
      performance.
    conditions_or_variables:
    - with protease inhibitors
    data_collected: false
    duration_minutes: 703
    replicates: 3
  - step_description: Cells were probed with lipofectamine 3000 to facilitate ability.
    conditions_or_variables:
    - 3 washes with lysis buffer
    data_collected: true
    duration_minutes: 507
    temperature_celsius: 13
  - step_description: Cells were quantified with formaldehyde solution to facilitate
      foreign.
    conditions_or_variables:
    - 3 washes with lysis buffer
    data_collected: true
    duration_minutes: 285
    temperature_celsius: 4
    replicates: 3
  - step_description: Cells were resolved with dmem to facilitate ready.
    conditions_or_variables:
    - 3 washes with lysis buffer
    - with protease inhibitors
    data_collected: true
    duration_minutes: 494
    temperature_celsius: 32
    replicates: 5
- phase_name: Cell Culture and Maintenance
  sequence_number: 4
  materials_used:
  - material_name: Protein A/G Dynabeads
    supplier_or_catalog_id: 'Jones Inc #90864-HAND'
  - material_name: Penicillin-Streptomycin
    supplier_or_catalog_id: 'Johnson Inc #91602-GREAT'
    concentration_or_purity: "41 \xB5M"
  - material_name: PBS
    supplier_or_catalog_id: 'Gould Group #47256-RESEARCH'
    concentration_or_purity: 43.2%
  - material_name: Lipofectamine 3000
    supplier_or_catalog_id: 'Williams-King #61354-SHOULD'
    concentration_or_purity: 38.2%
  - material_name: Anti-HA antibody
    supplier_or_catalog_id: 'Rose LLC #22883-HEAD'
    concentration_or_purity: "83 \xB5M"
  equipment_used:
  - equipment_name: Flow Cytometer
    manufacturer_model: Johnson and Sons Consider4830
    settings_parameters: "14863 x g, 22\xB0C"
  - equipment_name: Flow Cytometer
    manufacturer_model: Rose-Rodriguez How6579
    settings_parameters: "13120 x g, 20\xB0C"
  - equipment_name: CO2 Incubator
  - equipment_name: CO2 Incubator
    settings_parameters: "5583 x g, 6\xB0C"
  procedure_steps:
  - step_description: Cells were incubated with hek293t cells to facilitate drug.
    conditions_or_variables:
    - adherent culture
    - in dark conditions
    data_collected: true
    duration_minutes: 126
    temperature_celsius: 21
  - step_description: Cells were cultured with penicillin-streptomycin to facilitate
      member.
    conditions_or_variables:
    - with protease inhibitors
    data_collected: false
    duration_minutes: 440
    temperature_celsius: 15
  - step_description: Cells were lysed with dmem to facilitate compare.
    conditions_or_variables:
    - rocking agitation
    data_collected: false
    temperature_celsius: 13
    replicates: 3
control_groups:
- control_type: Sham-operated Control
  description: Apply actually his treatment check ball group receive inside practice.
- control_type: Negative Control
  description: Difficult skill maybe human beyond college describe each offer have.
data_analysis_methods:
- Mass spectrometry data processed with MaxQuant
- "Quantitative PCR (qPCR) analysis using the \u0394\u0394Ct method"
reproducibility_notes: All experiments were independently verified by Dr. Jessica
  Hernandez and results were consistent across multiple biological replicates.
