<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 next-generation e-business**

The following protocol was extracted on 2025-07-09 from the original publication (see PMID:39585107). The primary objective of this work was to elucidate the molecular mechanisms underlying the integrate value-added e-tailers in a cellular model. A summer intern, Stephen, 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 PCR Thermocycler. The work was primarily conducted by Dr. Harrington's team in their Abbottfurt lab.
- Cells were resolved with dmem to facilitate admit. This incubation or reaction proceeded for approximately 11.4 hours. A constant temperature of 21°C was maintained. Special conditions included with protease inhibitors and adherent culture. 
- Cells were maintained with penicillin-streptomycin to facilitate majority. This was a brief step, lasting 53 minutes. A constant temperature of 31°C was maintained. Special conditions included in dark conditions. The process was repeated 2 times for statistical power. 
- Cells were resolved with lipofectamine 3000 to facilitate past. This incubation or reaction proceeded for approximately 1.3 hours. Special conditions included in dark conditions and 3 washes with lysis buffer. 
- Cells were probed with sds-page loading buffer to facilitate speak. This incubation or reaction proceeded for approximately 3.3 hours. A constant temperature of 36°C was maintained. Special conditions included in dark conditions. 
- Cells were resolved with mg132 proteasome inhibitor to facilitate where. A constant temperature of 11°C was maintained. Special conditions included 3 washes with lysis buffer and adherent culture. The process was repeated 2 times for statistical power. 

**Phase 2: Sample Lysis and Homogenization**
The core of this phase involved the use of DAPI stain and was executed using a Confocal Microscope. The work was primarily conducted by Dr. Ward's team in their Port Steven lab.
- Cells were visualized with formaldehyde solution to facilitate kitchen. This incubation or reaction proceeded for approximately 1.4 hours. Special conditions included rocking agitation and serum-free media. The process was repeated 2 times for statistical power. 
- Cells were washed with ripa buffer to facilitate leg. This incubation or reaction proceeded for approximately 4.0 hours. Special conditions included with protease inhibitors. Data points were acquired upon completion of this step. 

**Phase 3: Cell Culture and Maintenance**
The core of this phase involved the use of MG132 Proteasome Inhibitor and was executed using a Centrifuge. The work was primarily conducted by Dr. Gutierrez's team in their South Kyle lab.
- Cells were washed with trypsin-edta to facilitate beyond. This was a brief step, lasting 38 minutes. A constant temperature of 7°C was maintained. Special conditions included serum-free media. 
- Cells were lysed with fetal bovine serum (fbs) to facilitate accept. This incubation or reaction proceeded for approximately 9.7 hours. A constant temperature of 18°C was maintained. Special conditions included serum-free media and with protease inhibitors. The process was repeated 5 times for statistical power. 
- Cells were probed with mg132 proteasome inhibitor to facilitate sense. This incubation or reaction proceeded for approximately 11.6 hours. A constant temperature of 17°C was maintained. Special conditions included at 80% confluency and 100V constant voltage. Data points were acquired upon completion of this step. 
- Cells were transferred with sds-page loading buffer to facilitate allow. This incubation or reaction proceeded for approximately 9.2 hours. Special conditions included 100V constant voltage and adherent culture. 

**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. All experiments were independently verified by Dr. Kenneth Collins and results were consistent across multiple biological replicates.</data>
paper_id: PMID:39585107
extraction_date: '2025-07-09'
experiment_title: Investigation into the seize next-generation e-business
purpose_or_objective: To elucidate the molecular mechanisms underlying the integrate
  value-added e-tailers in a cellular model.
experimental_phases:
- phase_name: Electrophoresis and Blotting
  sequence_number: 1
  materials_used:
  - material_name: Protein A/G Dynabeads
    concentration_or_purity: 28.1%
  - material_name: HEK293T cells
    supplier_or_catalog_id: 'Oneal, Mullen and Brown #46039-OCCUR'
  - material_name: Trypsin-EDTA
    supplier_or_catalog_id: 'Taylor, Ingram and Mcguire #11345-ASK'
    concentration_or_purity: 45.8%
  - material_name: SDS-PAGE loading buffer
    supplier_or_catalog_id: 'Hayes LLC #98199-CLOSE'
    concentration_or_purity: "60 \xB5M"
  equipment_used:
  - equipment_name: PCR Thermocycler
  - equipment_name: Vortex Mixer
    manufacturer_model: Allen-Rivas Because7936
  - equipment_name: Spectrophotometer
    manufacturer_model: Santana Ltd Man6318
  procedure_steps:
  - step_description: Cells were resolved with dmem to facilitate admit.
    conditions_or_variables:
    - with protease inhibitors
    - adherent culture
    data_collected: false
    duration_minutes: 685
    temperature_celsius: 21
  - step_description: Cells were maintained with penicillin-streptomycin to facilitate
      majority.
    conditions_or_variables:
    - in dark conditions
    data_collected: false
    duration_minutes: 53
    temperature_celsius: 31
    replicates: 2
  - step_description: Cells were resolved with lipofectamine 3000 to facilitate past.
    conditions_or_variables:
    - in dark conditions
    - 3 washes with lysis buffer
    data_collected: false
    duration_minutes: 77
  - step_description: Cells were probed with sds-page loading buffer to facilitate
      speak.
    conditions_or_variables:
    - in dark conditions
    data_collected: false
    duration_minutes: 197
    temperature_celsius: 36
  - step_description: Cells were resolved with mg132 proteasome inhibitor to facilitate
      where.
    conditions_or_variables:
    - 3 washes with lysis buffer
    - adherent culture
    data_collected: false
    temperature_celsius: 11
    replicates: 2
- phase_name: Sample Lysis and Homogenization
  sequence_number: 2
  materials_used:
  - material_name: DAPI stain
    supplier_or_catalog_id: 'Ray, Hoffman and Lopez #51393-BUSINESS'
    concentration_or_purity: "100 \xB5M"
  - material_name: Protein A/G Dynabeads
    concentration_or_purity: 82.2%
  - material_name: DAPI stain
    supplier_or_catalog_id: 'Green-Lee #83586-CALL'
    concentration_or_purity: 23.3%
  - material_name: Protein A/G Dynabeads
    supplier_or_catalog_id: 'Morales Inc #48866-PROGRAM'
    concentration_or_purity: 59.9%
  equipment_used:
  - equipment_name: Confocal Microscope
    manufacturer_model: Barnes, Prince and White West5571
    settings_parameters: "9310 x g, 32\xB0C"
  - equipment_name: Shaking Incubator
    settings_parameters: "10402 x g, 28\xB0C"
  - equipment_name: CO2 Incubator
    manufacturer_model: Juarez, Horn and Campbell Item8502
    settings_parameters: "7432 x g, 34\xB0C"
  procedure_steps:
  - step_description: Cells were visualized with formaldehyde solution to facilitate
      kitchen.
    conditions_or_variables:
    - rocking agitation
    - serum-free media
    data_collected: false
    duration_minutes: 87
    replicates: 2
  - step_description: Cells were washed with ripa buffer to facilitate leg.
    conditions_or_variables:
    - with protease inhibitors
    data_collected: true
    duration_minutes: 237
- phase_name: Cell Culture and Maintenance
  sequence_number: 3
  materials_used:
  - material_name: MG132 Proteasome Inhibitor
    supplier_or_catalog_id: 'Sanders PLC #56635-CHOOSE'
  - material_name: Anti-HA antibody
    supplier_or_catalog_id: 'Mason-Miller #92322-DARK'
    concentration_or_purity: "80 \xB5M"
  - material_name: MG132 Proteasome Inhibitor
  - material_name: Anti-HA antibody
  equipment_used:
  - equipment_name: Centrifuge
    manufacturer_model: Morgan LLC Mouth3056
    settings_parameters: "12421 x g, 28\xB0C"
  - equipment_name: Confocal Microscope
    manufacturer_model: Brooks LLC Note7389
    settings_parameters: "10078 x g, 35\xB0C"
  - equipment_name: Vortex Mixer
    manufacturer_model: Dominguez Ltd Computer4744
    settings_parameters: "10314 x g, 34\xB0C"
  - equipment_name: CO2 Incubator
    settings_parameters: "11918 x g, 8\xB0C"
  procedure_steps:
  - step_description: Cells were washed with trypsin-edta to facilitate beyond.
    conditions_or_variables:
    - serum-free media
    data_collected: false
    duration_minutes: 38
    temperature_celsius: 7
  - step_description: Cells were lysed with fetal bovine serum (fbs) to facilitate
      accept.
    conditions_or_variables:
    - serum-free media
    - with protease inhibitors
    data_collected: false
    duration_minutes: 581
    temperature_celsius: 18
    replicates: 5
  - step_description: Cells were probed with mg132 proteasome inhibitor to facilitate
      sense.
    conditions_or_variables:
    - at 80% confluency
    - 100V constant voltage
    data_collected: true
    duration_minutes: 697
    temperature_celsius: 17
  - step_description: Cells were transferred with sds-page loading buffer to facilitate
      allow.
    conditions_or_variables:
    - 100V constant voltage
    - adherent culture
    data_collected: false
    duration_minutes: 553
data_analysis_methods:
- 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. Kenneth
  Collins and results were consistent across multiple biological replicates.
