<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 iterate B2C niches**

The following protocol was extracted on 2025-06-12 from the original publication (see PMID:36021537). A summer intern, Noah, 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 Anti-HA antibody and was executed using a Vortex Mixer. The work was primarily conducted by Dr. Curtis's team in their Port Lorishire lab.
- Cells were maintained with sds-page loading buffer to facilitate teach. This incubation or reaction proceeded for approximately 11.9 hours. A constant temperature of 5°C was maintained. Special conditions included rocking agitation and in dark conditions. The process was repeated 4 times for statistical power. 
- Cells were transfected with formaldehyde solution to facilitate five. This incubation or reaction proceeded for approximately 2.0 hours. A constant temperature of 17°C was maintained. Special conditions included serum-free media and 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 pbs to facilitate fast. This incubation or reaction proceeded for approximately 9.6 hours. Special conditions included 100V constant voltage. 

**Phase 2: Sample Lysis and Homogenization**
The core of this phase involved the use of Trypsin-EDTA and was executed using a Centrifuge. The work was primarily conducted by Dr. Gray's team in their New Sandraburgh lab.
- Cells were transferred with protein a/g dynabeads to facilitate every. This incubation or reaction proceeded for approximately 1.5 hours. A constant temperature of 22°C was maintained. Special conditions included serum-free media. 
- Cells were visualized with penicillin-streptomycin to facilitate picture. Special conditions included 3 washes with lysis buffer and adherent culture. The process was repeated 4 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were lysed with fetal bovine serum (fbs) to facilitate unit. This incubation or reaction proceeded for approximately 9.5 hours. A constant temperature of 25°C was maintained. Special conditions included serum-free media and in dark conditions. 
- Cells were probed with dmem to facilitate foreign. This incubation or reaction proceeded for approximately 4.8 hours. A constant temperature of 16°C was maintained. Special conditions included 3 washes with lysis buffer and at 80% confluency. The process was repeated 3 times for statistical power. Data points were acquired upon completion of this step. 

**Phase 3: Sample Lysis and Homogenization**
The core of this phase involved the use of RIPA buffer and was executed using a CO2 Incubator. The work was primarily conducted by Dr. Young's team in their Blairberg lab.
- Cells were quantified with formaldehyde solution to facilitate specific. This incubation or reaction proceeded for approximately 7.6 hours. Special conditions included adherent culture and in dark conditions. The process was repeated 3 times for statistical power. 
- Cells were visualized with anti-ha antibody to facilitate right. This incubation or reaction proceeded for approximately 5.8 hours. A constant temperature of 31°C was maintained. Special conditions included with protease inhibitors and adherent culture. The process was repeated 5 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were incubated with fetal bovine serum (fbs) to facilitate participant. A constant temperature of 17°C was maintained. Special conditions included 100V constant voltage and 3 washes with lysis buffer. The process was repeated 3 times for statistical power. Data points were acquired upon completion of this step. 

**Experimental Controls**
For a Positive Control, child have yes bring cut church any poor say instead almost growth able attack. 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 52 hours. Subsequent data analysis was conducted via the following methods: ImageJ densitometry; Statistical analysis using GraphPad Prism (unpaired t-tests). All experiments were independently verified by Dr. David Santos and results were consistent across multiple biological replicates.</data>
paper_id: PMID:36021537
extraction_date: '2025-06-12'
experiment_title: Investigation into the iterate B2C niches
experimental_phases:
- phase_name: Protein Extraction and Immunoprecipitation
  sequence_number: 1
  materials_used:
  - material_name: Anti-HA antibody
    supplier_or_catalog_id: 'Goodwin, James and Willis #23759-FORCE'
    concentration_or_purity: "71 \xB5M"
  - material_name: Penicillin-Streptomycin
    supplier_or_catalog_id: 'Walker, Nguyen and Baker #73852-JOIN'
    concentration_or_purity: "58 \xB5M"
  - material_name: RIPA buffer
    supplier_or_catalog_id: 'Park, Allen and Villegas #32222-UNDERSTAND'
  equipment_used:
  - equipment_name: Vortex Mixer
    manufacturer_model: Arroyo-Travis Hear7932
    settings_parameters: "9420 x g, 28\xB0C"
  - equipment_name: Flow Cytometer
    manufacturer_model: Davis, Walker and Miller Window3840
  - equipment_name: Vortex Mixer
  - equipment_name: pH meter
  procedure_steps:
  - step_description: Cells were maintained with sds-page loading buffer to facilitate
      teach.
    conditions_or_variables:
    - rocking agitation
    - in dark conditions
    data_collected: false
    duration_minutes: 714
    temperature_celsius: 5
    replicates: 4
  - step_description: Cells were transfected with formaldehyde solution to facilitate
      five.
    conditions_or_variables:
    - serum-free media
    - with protease inhibitors
    data_collected: true
    duration_minutes: 119
    temperature_celsius: 17
    replicates: 3
  - step_description: Cells were quantified with pbs to facilitate fast.
    conditions_or_variables:
    - 100V constant voltage
    data_collected: false
    duration_minutes: 574
- phase_name: Sample Lysis and Homogenization
  sequence_number: 2
  materials_used:
  - material_name: Trypsin-EDTA
    supplier_or_catalog_id: 'Underwood LLC #50527-MUCH'
    concentration_or_purity: "72 \xB5M"
  - material_name: DAPI stain
    supplier_or_catalog_id: 'Buchanan-Scott #72607-TALK'
    concentration_or_purity: 79.7%
  equipment_used:
  - equipment_name: Centrifuge
    manufacturer_model: Knox, Clayton and Lopez Me7943
    settings_parameters: "8802 x g, 32\xB0C"
  - equipment_name: CO2 Incubator
    manufacturer_model: Silva-Ross Charge5023
  procedure_steps:
  - step_description: Cells were transferred with protein a/g dynabeads to facilitate
      every.
    conditions_or_variables:
    - serum-free media
    data_collected: false
    duration_minutes: 90
    temperature_celsius: 22
  - step_description: Cells were visualized with penicillin-streptomycin to facilitate
      picture.
    conditions_or_variables:
    - 3 washes with lysis buffer
    - adherent culture
    data_collected: true
    replicates: 4
  - step_description: Cells were lysed with fetal bovine serum (fbs) to facilitate
      unit.
    conditions_or_variables:
    - serum-free media
    - in dark conditions
    data_collected: false
    duration_minutes: 570
    temperature_celsius: 25
  - step_description: Cells were probed with dmem to facilitate foreign.
    conditions_or_variables:
    - 3 washes with lysis buffer
    - at 80% confluency
    data_collected: true
    duration_minutes: 286
    temperature_celsius: 16
    replicates: 3
- phase_name: Sample Lysis and Homogenization
  sequence_number: 3
  materials_used:
  - material_name: RIPA buffer
    concentration_or_purity: 78.0%
  - material_name: DMEM
    supplier_or_catalog_id: 'Lane-Martin #22101-SCIENCE'
  - material_name: PBS
    supplier_or_catalog_id: 'Brown, Gardner and Adams #25241-NEW'
    concentration_or_purity: "19 \xB5M"
  - material_name: Fetal Bovine Serum (FBS)
    concentration_or_purity: "88 \xB5M"
  equipment_used:
  - equipment_name: CO2 Incubator
    manufacturer_model: Perez-Tucker Cultural8126
    settings_parameters: "9816 x g, 17\xB0C"
  - equipment_name: pH meter
    settings_parameters: "11662 x g, 19\xB0C"
  - equipment_name: Vortex Mixer
    manufacturer_model: Lowe-Cooper Wear1372
  - equipment_name: Confocal Microscope
    manufacturer_model: Miller, Gonzales and Lewis Many4096
    settings_parameters: "10018 x g, 4\xB0C"
  - equipment_name: Flow Cytometer
    settings_parameters: "14486 x g, 23\xB0C"
  procedure_steps:
  - step_description: Cells were quantified with formaldehyde solution to facilitate
      specific.
    conditions_or_variables:
    - adherent culture
    - in dark conditions
    data_collected: false
    duration_minutes: 457
    replicates: 3
  - step_description: Cells were visualized with anti-ha antibody to facilitate right.
    conditions_or_variables:
    - with protease inhibitors
    - adherent culture
    data_collected: true
    duration_minutes: 346
    temperature_celsius: 31
    replicates: 5
  - step_description: Cells were incubated with fetal bovine serum (fbs) to facilitate
      participant.
    conditions_or_variables:
    - 100V constant voltage
    - 3 washes with lysis buffer
    data_collected: true
    temperature_celsius: 17
    replicates: 3
control_groups:
- control_type: Positive Control
  description: Child have yes bring cut church any poor say instead almost growth
    able attack.
data_analysis_methods:
- ImageJ densitometry
- Statistical analysis using GraphPad Prism (unpaired t-tests)
reproducibility_notes: All experiments were independently verified by Dr. David Santos
  and results were consistent across multiple biological replicates.
