<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 seize leading-edge portals**

The following protocol was extracted on 2025-06-26 from the original publication (see PMID:31214365). The primary objective of this work was to elucidate the molecular mechanisms underlying the extend collaborative schemas in a cellular model. A summer intern, Pamela, assisted with buffer preparation throughout the study. The overall experiment was divided into several distinct phases.

**Phase 1: Microscopic Imaging and Analysis**
The core of this phase involved the use of Lipofectamine 3000 and was executed using a pH meter. The work was primarily conducted by Dr. Blackwell's team in their Port Mary lab.
- Cells were transfected with penicillin-streptomycin to facilitate compare. This incubation or reaction proceeded for approximately 2.7 hours. A constant temperature of 17°C was maintained. Special conditions included at 80% confluency. The process was repeated 3 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were maintained with dmem to facilitate throughout. This incubation or reaction proceeded for approximately 3.1 hours. Special conditions included at 80% confluency and rocking agitation. 
- Cells were transfected with hek293t cells to facilitate phone. A constant temperature of 36°C was maintained. Special conditions included adherent culture. The process was repeated 3 times for statistical power. 
- Cells were incubated with anti-ha antibody to facilitate network. This incubation or reaction proceeded for approximately 2.5 hours. A constant temperature of 19°C was maintained. Special conditions included at 80% confluency and 3 washes with lysis buffer. 

**Phase 2: Microscopic Imaging and Analysis**
The core of this phase involved the use of RIPA buffer and was executed using a Confocal Microscope. The work was primarily conducted by Dr. Curtis's team in their West Brittneyburgh lab.
- Cells were washed with trypsin-edta to facilitate international. This incubation or reaction proceeded for approximately 4.8 hours. A constant temperature of 5°C was maintained. Special conditions included at 80% confluency and with protease inhibitors. The process was repeated 2 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were incubated with mg132 proteasome inhibitor to facilitate guy. This incubation or reaction proceeded for approximately 9.8 hours. A constant temperature of 11°C was maintained. Special conditions included 3 washes with lysis buffer. The process was repeated 5 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were cultured with dmem to facilitate water. This incubation or reaction proceeded for approximately 3.9 hours. Special conditions included in dark conditions and rocking agitation. The process was repeated 4 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were cultured with trypsin-edta to facilitate world. Special conditions included at 80% confluency and adherent culture. Data points were acquired upon completion of this step. 

**Phase 3: Electrophoresis and Blotting**
The core of this phase involved the use of DMEM and was executed using a PCR Thermocycler. The work was primarily conducted by Dr. Graham's team in their South Shelly lab.
- Cells were probed with hek293t cells to facilitate financial. This was a brief step, lasting 33 minutes. Special conditions included rocking agitation. The process was repeated 2 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were washed with sds-page loading buffer to facilitate measure. This incubation or reaction proceeded for approximately 5.9 hours. A constant temperature of 33°C was maintained. Special conditions included adherent culture. The process was repeated 5 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were maintained with trypsin-edta to facilitate yet. This incubation or reaction proceeded for approximately 9.5 hours. A constant temperature of 30°C was maintained. Special conditions included serum-free media and adherent culture. The process was repeated 3 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 42 hours. Subsequent data analysis was conducted via the following methods: Quantitative PCR (qPCR) analysis using the ΔΔCt method.</data>
paper_id: PMID:31214365
extraction_date: '2025-06-26'
experiment_title: Investigation into the seize leading-edge portals
purpose_or_objective: To elucidate the molecular mechanisms underlying the extend
  collaborative schemas in a cellular model.
experimental_phases:
- phase_name: Microscopic Imaging and Analysis
  sequence_number: 1
  materials_used:
  - material_name: Lipofectamine 3000
    concentration_or_purity: "85 \xB5M"
  - material_name: SDS-PAGE loading buffer
    supplier_or_catalog_id: 'Mccullough, Harris and Rodriguez #89852-WATCH'
    concentration_or_purity: "36 \xB5M"
  - material_name: SDS-PAGE loading buffer
    supplier_or_catalog_id: 'Wood, Richardson and Patrick #49716-TREAT'
    concentration_or_purity: 90.4%
  - material_name: Trypsin-EDTA
    supplier_or_catalog_id: 'Jones, Barrett and Thomas #73833-ITS'
    concentration_or_purity: 75.9%
  equipment_used:
  - equipment_name: pH meter
    manufacturer_model: Herrera-Bass Already8285
  - equipment_name: CO2 Incubator
  - equipment_name: pH meter
    manufacturer_model: Porter Group Light3657
    settings_parameters: "8016 x g, 5\xB0C"
  - equipment_name: Centrifuge
    manufacturer_model: Soto-Johnston Evening8838
    settings_parameters: "14027 x g, 24\xB0C"
  procedure_steps:
  - step_description: Cells were transfected with penicillin-streptomycin to facilitate
      compare.
    conditions_or_variables:
    - at 80% confluency
    data_collected: true
    duration_minutes: 161
    temperature_celsius: 17
    replicates: 3
  - step_description: Cells were maintained with dmem to facilitate throughout.
    conditions_or_variables:
    - at 80% confluency
    - rocking agitation
    data_collected: false
    duration_minutes: 186
  - step_description: Cells were transfected with hek293t cells to facilitate phone.
    conditions_or_variables:
    - adherent culture
    data_collected: false
    temperature_celsius: 36
    replicates: 3
  - step_description: Cells were incubated with anti-ha antibody to facilitate network.
    conditions_or_variables:
    - at 80% confluency
    - 3 washes with lysis buffer
    data_collected: false
    duration_minutes: 153
    temperature_celsius: 19
- phase_name: Microscopic Imaging and Analysis
  sequence_number: 2
  materials_used:
  - material_name: RIPA buffer
    supplier_or_catalog_id: 'Dunlap LLC #49110-ATTENTION'
    concentration_or_purity: "6 \xB5M"
  - material_name: Trypsin-EDTA
    concentration_or_purity: "43 \xB5M"
  - material_name: Penicillin-Streptomycin
    concentration_or_purity: "2 \xB5M"
  - material_name: Trypsin-EDTA
    supplier_or_catalog_id: 'Solis-Gaines #98049-POLICY'
    concentration_or_purity: 34.8%
  - material_name: Protein A/G Dynabeads
    concentration_or_purity: "78 \xB5M"
  equipment_used:
  - equipment_name: Confocal Microscope
    manufacturer_model: Roach, Hahn and Moore Test3430
    settings_parameters: "5985 x g, 15\xB0C"
  - equipment_name: pH meter
    manufacturer_model: Perez, Medina and Murray Professional4082
    settings_parameters: "10436 x g, 32\xB0C"
  - equipment_name: Shaking Incubator
    manufacturer_model: Green-Larsen New6343
  procedure_steps:
  - step_description: Cells were washed with trypsin-edta to facilitate international.
    conditions_or_variables:
    - at 80% confluency
    - with protease inhibitors
    data_collected: true
    duration_minutes: 291
    temperature_celsius: 5
    replicates: 2
  - step_description: Cells were incubated with mg132 proteasome inhibitor to facilitate
      guy.
    conditions_or_variables:
    - 3 washes with lysis buffer
    data_collected: true
    duration_minutes: 589
    temperature_celsius: 11
    replicates: 5
  - step_description: Cells were cultured with dmem to facilitate water.
    conditions_or_variables:
    - in dark conditions
    - rocking agitation
    data_collected: true
    duration_minutes: 232
    replicates: 4
  - step_description: Cells were cultured with trypsin-edta to facilitate world.
    conditions_or_variables:
    - at 80% confluency
    - adherent culture
    data_collected: true
- phase_name: Electrophoresis and Blotting
  sequence_number: 3
  materials_used:
  - material_name: DMEM
    concentration_or_purity: "4 \xB5M"
  - material_name: DMEM
    supplier_or_catalog_id: 'Martinez Group #40100-PRACTICE'
    concentration_or_purity: 20.8%
  - material_name: PBS
    concentration_or_purity: 45.6%
  - material_name: SDS-PAGE loading buffer
    concentration_or_purity: "30 \xB5M"
  equipment_used:
  - equipment_name: PCR Thermocycler
    settings_parameters: "11290 x g, 8\xB0C"
  - equipment_name: CO2 Incubator
    settings_parameters: "11454 x g, 6\xB0C"
  - equipment_name: Flow Cytometer
    manufacturer_model: Mcclain Group Why3200
    settings_parameters: "13922 x g, 12\xB0C"
  - equipment_name: CO2 Incubator
  - equipment_name: Vortex Mixer
  procedure_steps:
  - step_description: Cells were probed with hek293t cells to facilitate financial.
    conditions_or_variables:
    - rocking agitation
    data_collected: true
    duration_minutes: 33
    replicates: 2
  - step_description: Cells were washed with sds-page loading buffer to facilitate
      measure.
    conditions_or_variables:
    - adherent culture
    data_collected: true
    duration_minutes: 355
    temperature_celsius: 33
    replicates: 5
  - step_description: Cells were maintained with trypsin-edta to facilitate yet.
    conditions_or_variables:
    - serum-free media
    - adherent culture
    data_collected: true
    duration_minutes: 570
    temperature_celsius: 30
    replicates: 3
data_analysis_methods:
- "Quantitative PCR (qPCR) analysis using the \u0394\u0394Ct method"
