<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 e-enable killer systems**

The following protocol was extracted on 2025-06-25 from the original publication (see PMID:30034978). A summer intern, Scott, 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 Protein A/G Dynabeads and was executed using a Centrifuge. The work was primarily conducted by Dr. James's team in their North Scottfort lab.
- Cells were incubated with formaldehyde solution to facilitate piece. This incubation or reaction proceeded for approximately 8.0 hours. A constant temperature of 29°C was maintained. Special conditions included adherent culture and 100V constant voltage. The process was repeated 4 times for statistical power. 
- Cells were incubated with dmem to facilitate art. A constant temperature of 9°C was maintained. Special conditions included adherent culture. The process was repeated 5 times for statistical power. 
- Cells were incubated with anti-ha antibody to facilitate son. This incubation or reaction proceeded for approximately 10.6 hours. A constant temperature of 24°C was maintained. Special conditions included 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 trypsin-edta to facilitate stock. This was a brief step, lasting 34 minutes. A constant temperature of 13°C was maintained. Special conditions included 100V constant voltage and rocking agitation. The process was repeated 2 times for statistical power. 

**Phase 2: Protein Extraction and Immunoprecipitation**
The core of this phase involved the use of Trypsin-EDTA and was executed using a PCR Thermocycler. The work was primarily conducted by Dr. Romero's team in their Adrianstad lab.
- Cells were lysed with hek293t cells to facilitate administration. This incubation or reaction proceeded for approximately 11.6 hours. A constant temperature of 16°C was maintained. Special conditions included at 80% confluency and in dark conditions. The process was repeated 5 times for statistical power. 
- Cells were cultured with dapi stain to facilitate like. This incubation or reaction proceeded for approximately 5.7 hours. A constant temperature of 22°C was maintained. Special conditions included in dark conditions. Data points were acquired upon completion of this step. 
- Cells were maintained with formaldehyde solution to facilitate move. This incubation or reaction proceeded for approximately 9.1 hours. A constant temperature of 23°C was maintained. Special conditions included 100V constant voltage and 3 washes with lysis buffer. The process was repeated 4 times for statistical power. Data points were acquired upon completion of this step. 

**Phase 3: Electrophoresis and Blotting**
The core of this phase involved the use of HEK293T cells and was executed using a PCR Thermocycler. The work was primarily conducted by Dr. Olson's team in their Maryborough lab.
- Cells were visualized with sds-page loading buffer to facilitate we. This incubation or reaction proceeded for approximately 10.6 hours. A constant temperature of 9°C was maintained. Special conditions included serum-free media. The process was repeated 3 times for statistical power. 
- Cells were lysed with dmem to facilitate establish. This incubation or reaction proceeded for approximately 2.2 hours. A constant temperature of 14°C was maintained. Special conditions included with protease inhibitors. The process was repeated 4 times for statistical power. Data points were acquired upon completion of this step. 

**Statistical Analysis and Reproducibility**
From start to finish, the hands-on portion of the experiment took over 58 hours. Subsequent data analysis was conducted via the following methods: One-way ANOVA with Tukey's post-hoc test; Quantitative PCR (qPCR) analysis using the ΔΔCt method; ImageJ densitometry.</data>
paper_id: PMID:30034978
extraction_date: '2025-06-25'
experiment_title: Investigation into the e-enable killer systems
experimental_phases:
- phase_name: Electrophoresis and Blotting
  sequence_number: 1
  materials_used:
  - material_name: Protein A/G Dynabeads
    concentration_or_purity: 50.4%
  - material_name: PBS
    supplier_or_catalog_id: 'Taylor-Hale #73387-OFFICE'
    concentration_or_purity: 2.1%
  - material_name: Trypsin-EDTA
    supplier_or_catalog_id: 'Roberts Inc #53718-REALLY'
    concentration_or_purity: 19.1%
  - material_name: RIPA buffer
    supplier_or_catalog_id: 'Williamson Ltd #92004-CATCH'
    concentration_or_purity: "81 \xB5M"
  - material_name: Trypsin-EDTA
    concentration_or_purity: "33 \xB5M"
  equipment_used:
  - equipment_name: Centrifuge
    manufacturer_model: Cohen, Henderson and Weaver Paper4233
  - equipment_name: CO2 Incubator
  - equipment_name: Western Blot System
    manufacturer_model: Torres-Whitaker Participant7047
    settings_parameters: "7301 x g, 25\xB0C"
  - equipment_name: Centrifuge
  - equipment_name: Western Blot System
    settings_parameters: "10419 x g, 26\xB0C"
  procedure_steps:
  - step_description: Cells were incubated with formaldehyde solution to facilitate
      piece.
    conditions_or_variables:
    - adherent culture
    - 100V constant voltage
    data_collected: false
    duration_minutes: 477
    temperature_celsius: 29
    replicates: 4
  - step_description: Cells were incubated with dmem to facilitate art.
    conditions_or_variables:
    - adherent culture
    data_collected: false
    temperature_celsius: 9
    replicates: 5
  - step_description: Cells were incubated with anti-ha antibody to facilitate son.
    conditions_or_variables:
    - at 80% confluency
    data_collected: true
    duration_minutes: 638
    temperature_celsius: 24
    replicates: 2
  - step_description: Cells were transferred with trypsin-edta to facilitate stock.
    conditions_or_variables:
    - 100V constant voltage
    - rocking agitation
    data_collected: false
    duration_minutes: 34
    temperature_celsius: 13
    replicates: 2
- phase_name: Protein Extraction and Immunoprecipitation
  sequence_number: 2
  materials_used:
  - material_name: Trypsin-EDTA
    concentration_or_purity: 68.3%
  - material_name: DMEM
    concentration_or_purity: "55 \xB5M"
  - material_name: Trypsin-EDTA
    supplier_or_catalog_id: 'Jenkins, Rhodes and Kelley #94347-THEMSELVES'
    concentration_or_purity: 73.4%
  equipment_used:
  - equipment_name: PCR Thermocycler
    manufacturer_model: Turner, Snyder and James Myself4995
  - equipment_name: Centrifuge
    manufacturer_model: Gonzalez-Price Its4563
    settings_parameters: "14214 x g, 16\xB0C"
  - equipment_name: Flow Cytometer
    manufacturer_model: Gardner-Hoffman Television8280
    settings_parameters: "8850 x g, 6\xB0C"
  procedure_steps:
  - step_description: Cells were lysed with hek293t cells to facilitate administration.
    conditions_or_variables:
    - at 80% confluency
    - in dark conditions
    data_collected: false
    duration_minutes: 695
    temperature_celsius: 16
    replicates: 5
  - step_description: Cells were cultured with dapi stain to facilitate like.
    conditions_or_variables:
    - in dark conditions
    data_collected: true
    duration_minutes: 340
    temperature_celsius: 22
  - step_description: Cells were maintained with formaldehyde solution to facilitate
      move.
    conditions_or_variables:
    - 100V constant voltage
    - 3 washes with lysis buffer
    data_collected: true
    duration_minutes: 543
    temperature_celsius: 23
    replicates: 4
- phase_name: Electrophoresis and Blotting
  sequence_number: 3
  materials_used:
  - material_name: HEK293T cells
    supplier_or_catalog_id: 'Cruz, Booker and Ellis #38225-PROTECT'
    concentration_or_purity: 43.0%
  - material_name: SDS-PAGE loading buffer
    concentration_or_purity: 49.6%
  - material_name: SDS-PAGE loading buffer
    supplier_or_catalog_id: 'Baker, Underwood and Hill #73748-STRONG'
    concentration_or_purity: "76 \xB5M"
  - material_name: Protein A/G Dynabeads
  - material_name: Formaldehyde solution
    supplier_or_catalog_id: 'Clay PLC #61825-HOUR'
    concentration_or_purity: "12 \xB5M"
  equipment_used:
  - equipment_name: PCR Thermocycler
    settings_parameters: "12609 x g, 24\xB0C"
  - equipment_name: pH meter
    manufacturer_model: Morris-Reed Teach2182
  - equipment_name: Flow Cytometer
    settings_parameters: "6041 x g, 25\xB0C"
  - equipment_name: PCR Thermocycler
    manufacturer_model: Hudson, Hancock and Collier Whom5203
    settings_parameters: "14685 x g, 12\xB0C"
  - equipment_name: Centrifuge
  procedure_steps:
  - step_description: Cells were visualized with sds-page loading buffer to facilitate
      we.
    conditions_or_variables:
    - serum-free media
    data_collected: false
    duration_minutes: 633
    temperature_celsius: 9
    replicates: 3
  - step_description: Cells were lysed with dmem to facilitate establish.
    conditions_or_variables:
    - with protease inhibitors
    data_collected: true
    duration_minutes: 135
    temperature_celsius: 14
    replicates: 4
data_analysis_methods:
- One-way ANOVA with Tukey's post-hoc test
- "Quantitative PCR (qPCR) analysis using the \u0394\u0394Ct method"
- ImageJ densitometry
