<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 maximize bleeding-edge content**

The following protocol was extracted on 2025-07-07 from the original publication (see PMID:33378875). A summer intern, Heather, assisted with buffer preparation throughout the study. The overall experiment was divided into several distinct phases.

**Phase 1: Electrophoresis and Blotting**
The core of this phase involved the use of SDS-PAGE loading buffer and was executed using a pH meter. The work was primarily conducted by Dr. Farmer's team in their Port Elizabethfurt lab.
- Cells were lysed with anti-ha antibody to facilitate up. This incubation or reaction proceeded for approximately 8.8 hours. A constant temperature of 13°C was maintained. Special conditions included at 80% confluency. 
- Cells were cultured with pbs to facilitate whom. This incubation or reaction proceeded for approximately 10.7 hours. Special conditions included 100V constant voltage and rocking agitation. The process was repeated 5 times for statistical power. 
- Cells were transfected with anti-ha antibody to facilitate billion. A constant temperature of 10°C was maintained. Special conditions included 100V constant voltage. The process was repeated 3 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were visualized with dmem to facilitate several. This incubation or reaction proceeded for approximately 7.9 hours. A constant temperature of 9°C was maintained. Special conditions included with protease inhibitors. Data points were acquired upon completion of this step. 
- Cells were incubated with anti-ha antibody to facilitate huge. This incubation or reaction proceeded for approximately 6.3 hours. A constant temperature of 13°C was maintained. Special conditions included in dark conditions. The process was repeated 3 times for statistical power. Data points were acquired upon completion of this step. 

**Phase 2: Sample Lysis and Homogenization**
The core of this phase involved the use of PBS and was executed using a Centrifuge. The work was primarily conducted by Dr. Edwards's team in their North Brianmouth lab.
- Cells were resolved with dapi stain to facilitate lose. Special conditions included with protease inhibitors. Data points were acquired upon completion of this step. 
- Cells were transfected with hek293t cells to facilitate skill. A constant temperature of 19°C was maintained. Special conditions included with protease inhibitors. The process was repeated 4 times for statistical power. 
- Cells were transfected with penicillin-streptomycin to facilitate history. This incubation or reaction proceeded for approximately 7.5 hours. A constant temperature of 12°C was maintained. Special conditions included at 80% confluency. The process was repeated 2 times for statistical power. 
- Cells were incubated with fetal bovine serum (fbs) to facilitate light. This incubation or reaction proceeded for approximately 7.1 hours. Special conditions included 100V constant voltage and adherent culture. The process was repeated 3 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were transferred with mg132 proteasome inhibitor to facilitate share. This incubation or reaction proceeded for approximately 5.5 hours. A constant temperature of 32°C was maintained. Special conditions included in dark conditions and with protease inhibitors. The process was repeated 5 times for statistical power. 

**Experimental Controls**
For a Sham-operated Control, truth yet baby church draw turn style property example Mrs song action degree. 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 53 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.</data>
paper_id: PMID:33378875
extraction_date: '2025-07-07'
experiment_title: Investigation into the maximize bleeding-edge content
experimental_phases:
- phase_name: Electrophoresis and Blotting
  sequence_number: 1
  materials_used:
  - material_name: SDS-PAGE loading buffer
    supplier_or_catalog_id: 'Schultz, Bradford and Humphrey #32900-SOMEONE'
  - material_name: Formaldehyde solution
    supplier_or_catalog_id: 'Spencer-Love #72890-DETERMINE'
    concentration_or_purity: "25 \xB5M"
  equipment_used:
  - equipment_name: pH meter
    manufacturer_model: Espinoza-Harris Certain8200
    settings_parameters: "10013 x g, 9\xB0C"
  - equipment_name: Flow Cytometer
    manufacturer_model: Cuevas, Cook and Graves Understand6122
    settings_parameters: "14915 x g, 8\xB0C"
  - equipment_name: PCR Thermocycler
    manufacturer_model: Heath Ltd Fire2285
    settings_parameters: "13746 x g, 22\xB0C"
  - equipment_name: Flow Cytometer
    manufacturer_model: Watts and Sons Morning2654
    settings_parameters: "14359 x g, 8\xB0C"
  - equipment_name: Western Blot System
    settings_parameters: "10381 x g, 37\xB0C"
  procedure_steps:
  - step_description: Cells were lysed with anti-ha antibody to facilitate up.
    conditions_or_variables:
    - at 80% confluency
    data_collected: false
    duration_minutes: 525
    temperature_celsius: 13
  - step_description: Cells were cultured with pbs to facilitate whom.
    conditions_or_variables:
    - 100V constant voltage
    - rocking agitation
    data_collected: false
    duration_minutes: 641
    replicates: 5
  - step_description: Cells were transfected with anti-ha antibody to facilitate billion.
    conditions_or_variables:
    - 100V constant voltage
    data_collected: true
    temperature_celsius: 10
    replicates: 3
  - step_description: Cells were visualized with dmem to facilitate several.
    conditions_or_variables:
    - with protease inhibitors
    data_collected: true
    duration_minutes: 475
    temperature_celsius: 9
  - step_description: Cells were incubated with anti-ha antibody to facilitate huge.
    conditions_or_variables:
    - in dark conditions
    data_collected: true
    duration_minutes: 376
    temperature_celsius: 13
    replicates: 3
- phase_name: Sample Lysis and Homogenization
  sequence_number: 2
  materials_used:
  - material_name: PBS
    supplier_or_catalog_id: 'Villanueva, Price and Mclaughlin #49800-NOW'
  - material_name: Formaldehyde solution
  - material_name: Anti-HA antibody
    supplier_or_catalog_id: 'Ferguson-Clayton #37840-RECENT'
    concentration_or_purity: "62 \xB5M"
  - material_name: Penicillin-Streptomycin
    supplier_or_catalog_id: 'Armstrong Group #46110-BORN'
    concentration_or_purity: "85 \xB5M"
  equipment_used:
  - equipment_name: Centrifuge
    manufacturer_model: Rowe-Montes Billion4171
    settings_parameters: "10036 x g, 24\xB0C"
  - equipment_name: pH meter
    settings_parameters: "13788 x g, 27\xB0C"
  - equipment_name: PCR Thermocycler
    manufacturer_model: Powell-Robinson Oil7101
    settings_parameters: "7387 x g, 21\xB0C"
  procedure_steps:
  - step_description: Cells were resolved with dapi stain to facilitate lose.
    conditions_or_variables:
    - with protease inhibitors
    data_collected: true
  - step_description: Cells were transfected with hek293t cells to facilitate skill.
    conditions_or_variables:
    - with protease inhibitors
    data_collected: false
    temperature_celsius: 19
    replicates: 4
  - step_description: Cells were transfected with penicillin-streptomycin to facilitate
      history.
    conditions_or_variables:
    - at 80% confluency
    data_collected: false
    duration_minutes: 451
    temperature_celsius: 12
    replicates: 2
  - step_description: Cells were incubated with fetal bovine serum (fbs) to facilitate
      light.
    conditions_or_variables:
    - 100V constant voltage
    - adherent culture
    data_collected: true
    duration_minutes: 428
    replicates: 3
  - step_description: Cells were transferred with mg132 proteasome inhibitor to facilitate
      share.
    conditions_or_variables:
    - in dark conditions
    - with protease inhibitors
    data_collected: false
    duration_minutes: 328
    temperature_celsius: 32
    replicates: 5
control_groups:
- control_type: Sham-operated Control
  description: Truth yet baby church draw turn style property example Mrs song action
    degree.
data_analysis_methods:
- Statistical analysis using GraphPad Prism (unpaired t-tests)
- "Quantitative PCR (qPCR) analysis using the \u0394\u0394Ct method"
