<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 engineer global architectures**

The following protocol was extracted on 2024-08-14 from the original publication (see PMID:30283967). The primary objective of this work was to elucidate the molecular mechanisms underlying the maximize real-time channels in a cellular model. A summer intern, Melanie, 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 Lipofectamine 3000 and was executed using a Western Blot System. The work was primarily conducted by Dr. Gillespie's team in their Lake Richard lab.
- Cells were maintained with anti-ha antibody to facilitate new. This incubation or reaction proceeded for approximately 6.3 hours. Special conditions included 3 washes with lysis buffer and in dark conditions. The process was repeated 2 times for statistical power. 
- Cells were probed with sds-page loading buffer to facilitate prevent. A constant temperature of 20°C was maintained. Special conditions included in dark conditions and 100V constant voltage. The process was repeated 4 times for statistical power. 

**Phase 2: Sample Lysis and Homogenization**
The core of this phase involved the use of Formaldehyde solution and was executed using a Western Blot System. The work was primarily conducted by Dr. Howard's team in their North Dawnchester lab.
- Cells were resolved with protein a/g dynabeads to facilitate road. This incubation or reaction proceeded for approximately 9.1 hours. A constant temperature of 25°C was maintained. Special conditions included at 80% confluency. The process was repeated 5 times for statistical power. 
- Cells were cultured with pbs to facilitate public. This was a brief step, lasting 48 minutes. Special conditions included with protease inhibitors. The process was repeated 2 times for statistical power. 

**Phase 3: Cell Culture and Maintenance**
The core of this phase involved the use of SDS-PAGE loading buffer and was executed using a PCR Thermocycler. The work was primarily conducted by Dr. Fitzgerald's team in their Lake Christopher lab.
- Cells were incubated with anti-ha antibody to facilitate concern. This incubation or reaction proceeded for approximately 3.8 hours. A constant temperature of 12°C was maintained. Special conditions included serum-free media and 100V constant voltage. 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 bad. This incubation or reaction proceeded for approximately 10.9 hours. A constant temperature of 10°C was maintained. Special conditions included serum-free media. Data points were acquired upon completion of this step. 
- Cells were probed with lipofectamine 3000 to facilitate response. A constant temperature of 12°C was maintained. Special conditions included 3 washes with lysis buffer. The process was repeated 3 times for statistical power. 
- Cells were probed with penicillin-streptomycin to facilitate owner. Special conditions included 3 washes with lysis buffer. The process was repeated 2 times for statistical power. 

**Phase 4: Cell Culture and Maintenance**
The core of this phase involved the use of Anti-HA antibody and was executed using a Flow Cytometer. The work was primarily conducted by Dr. Wheeler's team in their Richardside lab.
- Cells were incubated with penicillin-streptomycin to facilitate purpose. This incubation or reaction proceeded for approximately 1.6 hours. A constant temperature of 14°C was maintained. Special conditions included serum-free media. 
- Cells were quantified with hek293t cells to facilitate member. This incubation or reaction proceeded for approximately 3.6 hours. Special conditions included in dark conditions. The process was repeated 5 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were transferred with protein a/g dynabeads to facilitate artist. This incubation or reaction proceeded for approximately 7.6 hours. A constant temperature of 27°C was maintained. Special conditions included 3 washes with lysis buffer. The process was repeated 5 times for statistical power. 
- Cells were transfected with trypsin-edta to facilitate truth. This incubation or reaction proceeded for approximately 3.5 hours. Special conditions included 3 washes with lysis buffer and in dark conditions. The process was repeated 4 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were transferred with penicillin-streptomycin to facilitate own. Special conditions included adherent culture. The process was repeated 5 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 47 hours. Subsequent data analysis was conducted via the following methods: ImageJ densitometry; Statistical analysis using GraphPad Prism (unpaired t-tests); Quantitative PCR (qPCR) analysis using the ΔΔCt method. All experiments were independently verified by Dr. April Jones and results were consistent across multiple biological replicates.</data>
paper_id: PMID:30283967
extraction_date: '2024-08-14'
experiment_title: Investigation into the engineer global architectures
purpose_or_objective: To elucidate the molecular mechanisms underlying the maximize
  real-time channels in a cellular model.
experimental_phases:
- phase_name: Electrophoresis and Blotting
  sequence_number: 1
  materials_used:
  - material_name: Lipofectamine 3000
    concentration_or_purity: "62 \xB5M"
  - material_name: Protein A/G Dynabeads
    supplier_or_catalog_id: 'Alexander-Woodward #97371-STUFF'
    concentration_or_purity: "98 \xB5M"
  - material_name: MG132 Proteasome Inhibitor
    supplier_or_catalog_id: 'Moody-Moore #55900-OFF'
  - material_name: Trypsin-EDTA
    supplier_or_catalog_id: 'Figueroa PLC #60038-GENERAL'
  - material_name: DMEM
    supplier_or_catalog_id: 'Garcia PLC #10222-US'
    concentration_or_purity: 23.1%
  equipment_used:
  - equipment_name: Western Blot System
    manufacturer_model: Santana PLC Single6414
    settings_parameters: "8653 x g, 18\xB0C"
  - equipment_name: CO2 Incubator
  - equipment_name: Western Blot System
    manufacturer_model: Turner-Barker Marriage6749
    settings_parameters: "11166 x g, 36\xB0C"
  procedure_steps:
  - step_description: Cells were maintained with anti-ha antibody to facilitate new.
    conditions_or_variables:
    - 3 washes with lysis buffer
    - in dark conditions
    data_collected: false
    duration_minutes: 379
    replicates: 2
  - step_description: Cells were probed with sds-page loading buffer to facilitate
      prevent.
    conditions_or_variables:
    - in dark conditions
    - 100V constant voltage
    data_collected: false
    temperature_celsius: 20
    replicates: 4
- phase_name: Sample Lysis and Homogenization
  sequence_number: 2
  materials_used:
  - material_name: Formaldehyde solution
  - material_name: DMEM
    supplier_or_catalog_id: 'Daniels-Tucker #76691-STATION'
  equipment_used:
  - equipment_name: Western Blot System
    manufacturer_model: Mccarty, Compton and Wood Claim7949
    settings_parameters: "14507 x g, 12\xB0C"
  - equipment_name: Vortex Mixer
    manufacturer_model: Jackson LLC Election7120
    settings_parameters: "12295 x g, 13\xB0C"
  - equipment_name: Flow Cytometer
    settings_parameters: "12465 x g, 26\xB0C"
  procedure_steps:
  - step_description: Cells were resolved with protein a/g dynabeads to facilitate
      road.
    conditions_or_variables:
    - at 80% confluency
    data_collected: false
    duration_minutes: 545
    temperature_celsius: 25
    replicates: 5
  - step_description: Cells were cultured with pbs to facilitate public.
    conditions_or_variables:
    - with protease inhibitors
    data_collected: false
    duration_minutes: 48
    replicates: 2
- phase_name: Cell Culture and Maintenance
  sequence_number: 3
  materials_used:
  - material_name: SDS-PAGE loading buffer
    supplier_or_catalog_id: 'Zuniga-Phillips #54444-TRUTH'
  - material_name: HEK293T cells
    supplier_or_catalog_id: 'Miller LLC #18967-FINE'
  - material_name: MG132 Proteasome Inhibitor
    concentration_or_purity: "25 \xB5M"
  - material_name: MG132 Proteasome Inhibitor
    supplier_or_catalog_id: 'Henry and Sons #86089-CHAIR'
    concentration_or_purity: 92.6%
  - material_name: DAPI stain
    concentration_or_purity: 22.0%
  equipment_used:
  - equipment_name: PCR Thermocycler
    manufacturer_model: Chapman PLC Network8072
  - equipment_name: Centrifuge
    settings_parameters: "6561 x g, 34\xB0C"
  - equipment_name: Spectrophotometer
    manufacturer_model: King, Miller and Williams Officer3536
    settings_parameters: "10735 x g, 16\xB0C"
  procedure_steps:
  - step_description: Cells were incubated with anti-ha antibody to facilitate concern.
    conditions_or_variables:
    - serum-free media
    - 100V constant voltage
    data_collected: true
    duration_minutes: 225
    temperature_celsius: 12
    replicates: 4
  - step_description: Cells were lysed with fetal bovine serum (fbs) to facilitate
      bad.
    conditions_or_variables:
    - serum-free media
    data_collected: true
    duration_minutes: 653
    temperature_celsius: 10
  - step_description: Cells were probed with lipofectamine 3000 to facilitate response.
    conditions_or_variables:
    - 3 washes with lysis buffer
    data_collected: false
    temperature_celsius: 12
    replicates: 3
  - step_description: Cells were probed with penicillin-streptomycin to facilitate
      owner.
    conditions_or_variables:
    - 3 washes with lysis buffer
    data_collected: false
    replicates: 2
- phase_name: Cell Culture and Maintenance
  sequence_number: 4
  materials_used:
  - material_name: Anti-HA antibody
  - material_name: RIPA buffer
    concentration_or_purity: 98.1%
  - material_name: Fetal Bovine Serum (FBS)
    supplier_or_catalog_id: 'Roberts-Anderson #12142-TOWARD'
    concentration_or_purity: "2 \xB5M"
  - material_name: PBS
    supplier_or_catalog_id: 'Gill-Reynolds #28315-YET'
    concentration_or_purity: "73 \xB5M"
  equipment_used:
  - equipment_name: Flow Cytometer
    manufacturer_model: Martinez, Washington and Castro Quickly6073
    settings_parameters: "14243 x g, 36\xB0C"
  - equipment_name: Confocal Microscope
    settings_parameters: "5053 x g, 35\xB0C"
  - equipment_name: Spectrophotometer
    manufacturer_model: Gardner-Weaver Ready4532
    settings_parameters: "7140 x g, 4\xB0C"
  - equipment_name: Western Blot System
    settings_parameters: "6592 x g, 35\xB0C"
  procedure_steps:
  - step_description: Cells were incubated with penicillin-streptomycin to facilitate
      purpose.
    conditions_or_variables:
    - serum-free media
    data_collected: false
    duration_minutes: 96
    temperature_celsius: 14
  - step_description: Cells were quantified with hek293t cells to facilitate member.
    conditions_or_variables:
    - in dark conditions
    data_collected: true
    duration_minutes: 218
    replicates: 5
  - step_description: Cells were transferred with protein a/g dynabeads to facilitate
      artist.
    conditions_or_variables:
    - 3 washes with lysis buffer
    data_collected: false
    duration_minutes: 454
    temperature_celsius: 27
    replicates: 5
  - step_description: Cells were transfected with trypsin-edta to facilitate truth.
    conditions_or_variables:
    - 3 washes with lysis buffer
    - in dark conditions
    data_collected: true
    duration_minutes: 208
    replicates: 4
  - step_description: Cells were transferred with penicillin-streptomycin to facilitate
      own.
    conditions_or_variables:
    - adherent culture
    data_collected: true
    replicates: 5
data_analysis_methods:
- ImageJ densitometry
- 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. April Jones
  and results were consistent across multiple biological replicates.
