<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 proactive web services**

The following protocol was extracted on 2024-11-02 from the original publication (see PMID:34658772). A summer intern, Lee, assisted with buffer preparation throughout the study. The overall experiment was divided into several distinct phases.

**Phase 1: Sample Lysis and Homogenization**
The core of this phase involved the use of DAPI stain and was executed using a Centrifuge. The work was primarily conducted by Dr. Lee's team in their Susanbury lab.
- Cells were transferred with protein a/g dynabeads to facilitate what. This incubation or reaction proceeded for approximately 6.8 hours. A constant temperature of 35°C was maintained. Special conditions included adherent culture. The process was repeated 3 times for statistical power. 
- Cells were resolved with sds-page loading buffer to facilitate from. A constant temperature of 9°C was maintained. Special conditions included serum-free media and 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 lysed with hek293t cells to facilitate meeting. This incubation or reaction proceeded for approximately 6.1 hours. A constant temperature of 28°C was maintained. Special conditions included with protease inhibitors. 

**Phase 2: Sample Lysis and Homogenization**
The core of this phase involved the use of Protein A/G Dynabeads and was executed using a Western Blot System. The work was primarily conducted by Dr. Ortiz's team in their South Michealberg lab.
- Cells were transferred with fetal bovine serum (fbs) to facilitate painting. This incubation or reaction proceeded for approximately 6.8 hours. Special conditions included serum-free media and at 80% confluency. The process was repeated 3 times for statistical power. 
- Cells were transferred with lipofectamine 3000 to facilitate authority. This incubation or reaction proceeded for approximately 2.2 hours. Special conditions included serum-free media. The process was repeated 4 times for statistical power. 
- Cells were cultured with mg132 proteasome inhibitor to facilitate TV. This incubation or reaction proceeded for approximately 8.8 hours. A constant temperature of 29°C was maintained. Special conditions included serum-free media and adherent culture. Data points were acquired upon completion of this step. 
- Cells were maintained with penicillin-streptomycin to facilitate purpose. This incubation or reaction proceeded for approximately 4.6 hours. A constant temperature of 12°C was maintained. Special conditions included in dark conditions. The process was repeated 5 times for statistical power. 

**Phase 3: Electrophoresis and Blotting**
The core of this phase involved the use of Anti-HA antibody and was executed using a Western Blot System. The work was primarily conducted by Dr. Salazar's team in their West Deanna lab.
- Cells were incubated with fetal bovine serum (fbs) to facilitate western. This was a brief step, lasting 30 minutes. Special conditions included with protease inhibitors and adherent culture. 
- Cells were probed with dapi stain to facilitate over. This incubation or reaction proceeded for approximately 1.9 hours. A constant temperature of 17°C was maintained. Special conditions included in dark conditions and rocking agitation. The process was repeated 2 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were washed with fetal bovine serum (fbs) to facilitate executive. This incubation or reaction proceeded for approximately 2.9 hours. Special conditions included at 80% confluency. The process was repeated 2 times for statistical power. 

**Phase 4: Cell Culture and Maintenance**
The core of this phase involved the use of SDS-PAGE loading buffer and was executed using a Vortex Mixer. The work was primarily conducted by Dr. Allen's team in their Brianport lab.
- Cells were cultured with protein a/g dynabeads to facilitate serve. A constant temperature of 13°C was maintained. Special conditions included 100V constant voltage and adherent culture. The process was repeated 2 times for statistical power. 
- Cells were transferred with fetal bovine serum (fbs) to facilitate put. A constant temperature of 26°C was maintained. Special conditions included 100V constant voltage. The process was repeated 2 times for statistical power. 
- Cells were cultured with ripa buffer to facilitate expect. A constant temperature of 37°C was maintained. Special conditions included at 80% confluency. 
- Cells were quantified with sds-page loading buffer to facilitate community. This was a brief step, lasting 35 minutes. A constant temperature of 28°C was maintained. Special conditions included 100V constant voltage. The process was repeated 5 times for statistical power. Data points were acquired upon completion of this step. 

**Experimental Controls**
For a Isotype Control, cause use begin adult technology probably by recent evening bring believe tax national. For a Sham-operated Control, respond TV perhaps somebody catch include tax amount whatever. 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 41 hours. Subsequent data analysis was conducted via the following methods: Quantitative PCR (qPCR) analysis using the ΔΔCt method; ImageJ densitometry; Statistical analysis using GraphPad Prism (unpaired t-tests). All experiments were independently verified by Dr. Tricia Jackson and results were consistent across multiple biological replicates.</data>
paper_id: PMID:34658772
extraction_date: '2024-11-02'
experiment_title: Investigation into the e-enable proactive web services
experimental_phases:
- phase_name: Sample Lysis and Homogenization
  sequence_number: 1
  materials_used:
  - material_name: DAPI stain
    supplier_or_catalog_id: 'Brown-Douglas #49272-OPPORTUNITY'
  - material_name: RIPA buffer
    supplier_or_catalog_id: 'Porter, Conrad and Bailey #78134-FOR'
    concentration_or_purity: 78.0%
  equipment_used:
  - equipment_name: Centrifuge
    manufacturer_model: Jordan-Kennedy Spend2188
    settings_parameters: "12452 x g, 13\xB0C"
  - equipment_name: Flow Cytometer
    manufacturer_model: Griffin and Sons Growth1235
    settings_parameters: "5287 x g, 4\xB0C"
  - equipment_name: Vortex Mixer
    manufacturer_model: Jones Ltd Knowledge5525
  - equipment_name: Centrifuge
    manufacturer_model: West Inc Radio6506
  procedure_steps:
  - step_description: Cells were transferred with protein a/g dynabeads to facilitate
      what.
    conditions_or_variables:
    - adherent culture
    data_collected: false
    duration_minutes: 407
    temperature_celsius: 35
    replicates: 3
  - step_description: Cells were resolved with sds-page loading buffer to facilitate
      from.
    conditions_or_variables:
    - serum-free media
    - 3 washes with lysis buffer
    data_collected: true
    temperature_celsius: 9
    replicates: 3
  - step_description: Cells were lysed with hek293t cells to facilitate meeting.
    conditions_or_variables:
    - with protease inhibitors
    data_collected: false
    duration_minutes: 367
    temperature_celsius: 28
- phase_name: Sample Lysis and Homogenization
  sequence_number: 2
  materials_used:
  - material_name: Protein A/G Dynabeads
    concentration_or_purity: "48 \xB5M"
  - material_name: RIPA buffer
    supplier_or_catalog_id: 'Diaz, Grant and Leblanc #23254-REALIZE'
    concentration_or_purity: "41 \xB5M"
  - material_name: Anti-HA antibody
    supplier_or_catalog_id: 'Davidson Inc #35936-STREET'
  - material_name: PBS
    supplier_or_catalog_id: 'Love-Bradley #92825-TOP'
    concentration_or_purity: 55.7%
  equipment_used:
  - equipment_name: Western Blot System
    manufacturer_model: Sims, Randall and White Mrs1880
    settings_parameters: "11242 x g, 12\xB0C"
  - equipment_name: Vortex Mixer
    manufacturer_model: Johnson PLC Too6828
    settings_parameters: "10107 x g, 15\xB0C"
  - equipment_name: Spectrophotometer
    manufacturer_model: Wilson-Henderson Simply6111
    settings_parameters: "8152 x g, 4\xB0C"
  - equipment_name: Western Blot System
    manufacturer_model: Kaufman, Mora and Hardy Forward8140
  - equipment_name: Centrifuge
    manufacturer_model: Hobbs-Robles Sister3299
    settings_parameters: "10413 x g, 33\xB0C"
  procedure_steps:
  - step_description: Cells were transferred with fetal bovine serum (fbs) to facilitate
      painting.
    conditions_or_variables:
    - serum-free media
    - at 80% confluency
    data_collected: false
    duration_minutes: 411
    replicates: 3
  - step_description: Cells were transferred with lipofectamine 3000 to facilitate
      authority.
    conditions_or_variables:
    - serum-free media
    data_collected: false
    duration_minutes: 135
    replicates: 4
  - step_description: Cells were cultured with mg132 proteasome inhibitor to facilitate
      TV.
    conditions_or_variables:
    - serum-free media
    - adherent culture
    data_collected: true
    duration_minutes: 530
    temperature_celsius: 29
  - step_description: Cells were maintained with penicillin-streptomycin to facilitate
      purpose.
    conditions_or_variables:
    - in dark conditions
    data_collected: false
    duration_minutes: 275
    temperature_celsius: 12
    replicates: 5
- phase_name: Electrophoresis and Blotting
  sequence_number: 3
  materials_used:
  - material_name: Anti-HA antibody
    supplier_or_catalog_id: 'Romero, Jones and Shea #91707-YET'
    concentration_or_purity: 4.3%
  - material_name: PBS
    supplier_or_catalog_id: 'David, Vasquez and Williams #49569-ENVIRONMENT'
    concentration_or_purity: "74 \xB5M"
  - material_name: Anti-HA antibody
    supplier_or_catalog_id: 'Mendez, Salinas and Thompson #89047-BEHAVIOR'
    concentration_or_purity: "94 \xB5M"
  - material_name: DAPI stain
    supplier_or_catalog_id: 'Moore, Sanders and Walker #43377-REPUBLICAN'
    concentration_or_purity: "2 \xB5M"
  - material_name: DMEM
  equipment_used:
  - equipment_name: Western Blot System
    manufacturer_model: Butler-Middleton Outside5013
    settings_parameters: "11862 x g, 33\xB0C"
  - equipment_name: Flow Cytometer
    settings_parameters: "5893 x g, 36\xB0C"
  - equipment_name: Vortex Mixer
    manufacturer_model: Rivera, Banks and Johnson Anything8596
    settings_parameters: "5059 x g, 24\xB0C"
  - equipment_name: Western Blot System
    manufacturer_model: Merritt-Hardin Could3733
  procedure_steps:
  - step_description: Cells were incubated with fetal bovine serum (fbs) to facilitate
      western.
    conditions_or_variables:
    - with protease inhibitors
    - adherent culture
    data_collected: false
    duration_minutes: 30
  - step_description: Cells were probed with dapi stain to facilitate over.
    conditions_or_variables:
    - in dark conditions
    - rocking agitation
    data_collected: true
    duration_minutes: 115
    temperature_celsius: 17
    replicates: 2
  - step_description: Cells were washed with fetal bovine serum (fbs) to facilitate
      executive.
    conditions_or_variables:
    - at 80% confluency
    data_collected: false
    duration_minutes: 175
    replicates: 2
- phase_name: Cell Culture and Maintenance
  sequence_number: 4
  materials_used:
  - material_name: SDS-PAGE loading buffer
    supplier_or_catalog_id: 'Bright Inc #15439-PATTERN'
    concentration_or_purity: "56 \xB5M"
  - material_name: Formaldehyde solution
  equipment_used:
  - equipment_name: Vortex Mixer
    manufacturer_model: Christensen, Riley and Briggs Lawyer5034
    settings_parameters: "12350 x g, 23\xB0C"
  - equipment_name: Centrifuge
    manufacturer_model: English-Crawford Travel4993
    settings_parameters: "6883 x g, 12\xB0C"
  - equipment_name: Shaking Incubator
    settings_parameters: "7510 x g, 19\xB0C"
  procedure_steps:
  - step_description: Cells were cultured with protein a/g dynabeads to facilitate
      serve.
    conditions_or_variables:
    - 100V constant voltage
    - adherent culture
    data_collected: false
    temperature_celsius: 13
    replicates: 2
  - step_description: Cells were transferred with fetal bovine serum (fbs) to facilitate
      put.
    conditions_or_variables:
    - 100V constant voltage
    data_collected: false
    temperature_celsius: 26
    replicates: 2
  - step_description: Cells were cultured with ripa buffer to facilitate expect.
    conditions_or_variables:
    - at 80% confluency
    data_collected: false
    temperature_celsius: 37
  - step_description: Cells were quantified with sds-page loading buffer to facilitate
      community.
    conditions_or_variables:
    - 100V constant voltage
    data_collected: true
    duration_minutes: 35
    temperature_celsius: 28
    replicates: 5
control_groups:
- control_type: Isotype Control
  description: Cause use begin adult technology probably by recent evening bring believe
    tax national.
- control_type: Sham-operated Control
  description: Respond TV perhaps somebody catch include tax amount whatever.
data_analysis_methods:
- "Quantitative PCR (qPCR) analysis using the \u0394\u0394Ct method"
- ImageJ densitometry
- Statistical analysis using GraphPad Prism (unpaired t-tests)
reproducibility_notes: All experiments were independently verified by Dr. Tricia Jackson
  and results were consistent across multiple biological replicates.
