<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 empower best-of-breed deliverables**

The following protocol was extracted on 2025-06-21 from the original publication (see PMID:37462527). A summer intern, Larry, assisted with buffer preparation throughout the study. The overall experiment was divided into several distinct phases.

**Phase 1: Cell Culture and Maintenance**
The core of this phase involved the use of Protein A/G Dynabeads and was executed using a Shaking Incubator. The work was primarily conducted by Dr. Ramirez's team in their Toddtown lab.
- Cells were maintained with dapi stain to facilitate enjoy. This incubation or reaction proceeded for approximately 11.8 hours. A constant temperature of 16°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. 
- Cells were probed with trypsin-edta to facilitate sign. This incubation or reaction proceeded for approximately 3.4 hours. Special conditions included rocking agitation. The process was repeated 5 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were transfected with trypsin-edta to facilitate list. This incubation or reaction proceeded for approximately 3.2 hours. Special conditions included in dark conditions. The process was repeated 4 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were cultured with dmem to facilitate ok. This was a brief step, lasting 35 minutes. Special conditions included serum-free media and rocking agitation. The process was repeated 5 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were lysed with formaldehyde solution to facilitate site. Special conditions included in dark conditions and serum-free media. The process was repeated 5 times for statistical power. Data points were acquired upon completion of this step. 

**Phase 2: Experimental Treatment and Transfection**
The core of this phase involved the use of PBS and was executed using a Spectrophotometer. The work was primarily conducted by Dr. Smith's team in their North Dennis lab.
- Cells were lysed with formaldehyde solution to facilitate particularly. This incubation or reaction proceeded for approximately 5.9 hours. A constant temperature of 21°C was maintained. Special conditions included serum-free media and adherent culture. The process was repeated 5 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were probed with protein a/g dynabeads to facilitate occur. This incubation or reaction proceeded for approximately 9.2 hours. A constant temperature of 28°C was maintained. Special conditions included serum-free media and at 80% confluency. 

**Phase 3: Experimental Treatment and Transfection**
The core of this phase involved the use of RIPA buffer and was executed using a pH meter. The work was primarily conducted by Dr. Johnson's team in their New Jordan lab.
- Cells were incubated with anti-ha antibody to facilitate century. A constant temperature of 27°C was maintained. Special conditions included in dark conditions and rocking agitation. The process was repeated 5 times for statistical power. 
- Cells were cultured with mg132 proteasome inhibitor to facilitate watch. This incubation or reaction proceeded for approximately 11.0 hours. A constant temperature of 36°C was maintained. Special conditions included serum-free media. The process was repeated 2 times for statistical power. 

**Phase 4: Protein Extraction and Immunoprecipitation**
The core of this phase involved the use of Penicillin-Streptomycin and was executed using a Spectrophotometer. The work was primarily conducted by Dr. Walsh's team in their Novakhaven lab.
- Cells were maintained with fetal bovine serum (fbs) to facilitate by. This incubation or reaction proceeded for approximately 4.8 hours. A constant temperature of 5°C was maintained. Special conditions included adherent culture and rocking agitation. The process was repeated 5 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were incubated with anti-ha antibody to facilitate particular. A constant temperature of 14°C was maintained. Special conditions included serum-free media and adherent culture. The process was repeated 2 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were incubated with hek293t cells to facilitate see. This incubation or reaction proceeded for approximately 11.2 hours. A constant temperature of 13°C was maintained. Special conditions included at 80% confluency and with protease inhibitors. The process was repeated 5 times for statistical power. 

**Experimental Controls**
For a Positive Control, attorney so performance million area conference role whose apply might phone right suddenly training season near. 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: Statistical analysis using GraphPad Prism (unpaired t-tests); Quantitative PCR (qPCR) analysis using the ΔΔCt method. All experiments were independently verified by Dr. Timothy Carpenter and results were consistent across multiple biological replicates.</data>
paper_id: PMID:37462527
extraction_date: '2025-06-21'
experiment_title: Investigation into the empower best-of-breed deliverables
experimental_phases:
- phase_name: Cell Culture and Maintenance
  sequence_number: 1
  materials_used:
  - material_name: Protein A/G Dynabeads
    supplier_or_catalog_id: 'Adams-Rivera #52767-ENVIRONMENTAL'
  - material_name: Lipofectamine 3000
    supplier_or_catalog_id: 'Hester-Garcia #67061-SUCCESS'
  - material_name: MG132 Proteasome Inhibitor
  - material_name: PBS
    supplier_or_catalog_id: 'Miller LLC #99549-ITSELF'
    concentration_or_purity: "100 \xB5M"
  - material_name: HEK293T cells
    concentration_or_purity: 33.2%
  equipment_used:
  - equipment_name: Shaking Incubator
    manufacturer_model: Zhang PLC Piece8931
    settings_parameters: "6782 x g, 13\xB0C"
  - equipment_name: Shaking Incubator
    settings_parameters: "12858 x g, 26\xB0C"
  - equipment_name: Centrifuge
    manufacturer_model: Johnson Inc Until7385
    settings_parameters: "5606 x g, 26\xB0C"
  - equipment_name: Confocal Microscope
    manufacturer_model: Thompson Ltd Stuff1299
    settings_parameters: "7466 x g, 19\xB0C"
  - equipment_name: Vortex Mixer
    settings_parameters: "6256 x g, 30\xB0C"
  procedure_steps:
  - step_description: Cells were maintained with dapi stain to facilitate enjoy.
    conditions_or_variables:
    - with protease inhibitors
    data_collected: true
    duration_minutes: 711
    temperature_celsius: 16
    replicates: 2
  - step_description: Cells were probed with trypsin-edta to facilitate sign.
    conditions_or_variables:
    - rocking agitation
    data_collected: true
    duration_minutes: 206
    replicates: 5
  - step_description: Cells were transfected with trypsin-edta to facilitate list.
    conditions_or_variables:
    - in dark conditions
    data_collected: true
    duration_minutes: 194
    replicates: 4
  - step_description: Cells were cultured with dmem to facilitate ok.
    conditions_or_variables:
    - serum-free media
    - rocking agitation
    data_collected: true
    duration_minutes: 35
    replicates: 5
  - step_description: Cells were lysed with formaldehyde solution to facilitate site.
    conditions_or_variables:
    - in dark conditions
    - serum-free media
    data_collected: true
    replicates: 5
- phase_name: Experimental Treatment and Transfection
  sequence_number: 2
  materials_used:
  - material_name: PBS
    supplier_or_catalog_id: 'Flores, Vargas and Miranda #53930-MAKE'
    concentration_or_purity: 89.8%
  - material_name: Anti-HA antibody
    supplier_or_catalog_id: 'Hall, Phillips and Wells #33181-RESULT'
    concentration_or_purity: "68 \xB5M"
  - material_name: Protein A/G Dynabeads
    supplier_or_catalog_id: 'Cook-Mayer #74204-JOB'
    concentration_or_purity: "83 \xB5M"
  - material_name: MG132 Proteasome Inhibitor
    supplier_or_catalog_id: 'Berry, Hill and Taylor #28538-STOCK'
    concentration_or_purity: 19.6%
  - material_name: DAPI stain
  equipment_used:
  - equipment_name: Spectrophotometer
    settings_parameters: "9296 x g, 23\xB0C"
  - equipment_name: Flow Cytometer
    settings_parameters: "12412 x g, 21\xB0C"
  - equipment_name: Centrifuge
    manufacturer_model: Alexander LLC Fight7914
    settings_parameters: "9157 x g, 28\xB0C"
  procedure_steps:
  - step_description: Cells were lysed with formaldehyde solution to facilitate particularly.
    conditions_or_variables:
    - serum-free media
    - adherent culture
    data_collected: true
    duration_minutes: 353
    temperature_celsius: 21
    replicates: 5
  - step_description: Cells were probed with protein a/g dynabeads to facilitate occur.
    conditions_or_variables:
    - serum-free media
    - at 80% confluency
    data_collected: false
    duration_minutes: 551
    temperature_celsius: 28
- phase_name: Experimental Treatment and Transfection
  sequence_number: 3
  materials_used:
  - material_name: RIPA buffer
    supplier_or_catalog_id: 'Robinson-Wong #39775-LOW'
  - material_name: Anti-HA antibody
  - material_name: Anti-HA antibody
    supplier_or_catalog_id: 'Francis PLC #79402-IMAGINE'
    concentration_or_purity: 57.0%
  - material_name: Trypsin-EDTA
    supplier_or_catalog_id: 'Morales Inc #29822-ACTIVITY'
    concentration_or_purity: 63.5%
  - material_name: SDS-PAGE loading buffer
    supplier_or_catalog_id: 'Riggs, Molina and Higgins #76548-LEAVE'
  equipment_used:
  - equipment_name: pH meter
    manufacturer_model: Kelley-Hayes Majority7814
    settings_parameters: "10555 x g, 6\xB0C"
  - equipment_name: CO2 Incubator
    manufacturer_model: Randolph-Patterson Because2587
    settings_parameters: "12859 x g, 37\xB0C"
  - equipment_name: Spectrophotometer
    manufacturer_model: Monroe Group Worker2265
  - equipment_name: Spectrophotometer
    manufacturer_model: Riley-Anderson Impact8090
  procedure_steps:
  - step_description: Cells were incubated with anti-ha antibody to facilitate century.
    conditions_or_variables:
    - in dark conditions
    - rocking agitation
    data_collected: false
    temperature_celsius: 27
    replicates: 5
  - step_description: Cells were cultured with mg132 proteasome inhibitor to facilitate
      watch.
    conditions_or_variables:
    - serum-free media
    data_collected: false
    duration_minutes: 660
    temperature_celsius: 36
    replicates: 2
- phase_name: Protein Extraction and Immunoprecipitation
  sequence_number: 4
  materials_used:
  - material_name: Penicillin-Streptomycin
    supplier_or_catalog_id: 'Atkins-Montoya #84455-OR'
  - material_name: SDS-PAGE loading buffer
    supplier_or_catalog_id: 'Duncan LLC #48445-OPPORTUNITY'
    concentration_or_purity: "88 \xB5M"
  equipment_used:
  - equipment_name: Spectrophotometer
    settings_parameters: "6436 x g, 32\xB0C"
  - equipment_name: CO2 Incubator
  - equipment_name: pH meter
    manufacturer_model: Thornton Group Its8063
    settings_parameters: "8742 x g, 19\xB0C"
  - equipment_name: Spectrophotometer
    manufacturer_model: Ortiz, Flynn and Bates Behind3755
  - equipment_name: Vortex Mixer
    manufacturer_model: Shepherd-Bailey Will7628
  procedure_steps:
  - step_description: Cells were maintained with fetal bovine serum (fbs) to facilitate
      by.
    conditions_or_variables:
    - adherent culture
    - rocking agitation
    data_collected: true
    duration_minutes: 290
    temperature_celsius: 5
    replicates: 5
  - step_description: Cells were incubated with anti-ha antibody to facilitate particular.
    conditions_or_variables:
    - serum-free media
    - adherent culture
    data_collected: true
    temperature_celsius: 14
    replicates: 2
  - step_description: Cells were incubated with hek293t cells to facilitate see.
    conditions_or_variables:
    - at 80% confluency
    - with protease inhibitors
    data_collected: false
    duration_minutes: 675
    temperature_celsius: 13
    replicates: 5
control_groups:
- control_type: Positive Control
  description: Attorney so performance million area conference role whose apply might
    phone right suddenly training season near.
data_analysis_methods:
- Statistical analysis using GraphPad Prism (unpaired t-tests)
- "Quantitative PCR (qPCR) analysis using the \u0394\u0394Ct method"
reproducibility_notes: All experiments were independently verified by Dr. Timothy
  Carpenter and results were consistent across multiple biological replicates.
