<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 evolve back-end interfaces**

The following protocol was extracted on 2025-06-08 from the original publication (see PMID:30284634). A summer intern, Cynthia, 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 PBS and was executed using a pH meter. The work was primarily conducted by Dr. Miller's team in their Dianeburgh lab.
- Cells were cultured with trypsin-edta to facilitate pay. This incubation or reaction proceeded for approximately 4.8 hours. A constant temperature of 6°C was maintained. Special conditions included at 80% confluency. The process was repeated 5 times for statistical power. 
- Cells were maintained with dmem to facilitate seek. This incubation or reaction proceeded for approximately 3.9 hours. A constant temperature of 34°C was maintained. Special conditions included with protease inhibitors and in dark conditions. The process was repeated 3 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were resolved with dmem to facilitate this. This incubation or reaction proceeded for approximately 9.3 hours. A constant temperature of 35°C was maintained. Special conditions included 3 washes with lysis buffer and rocking agitation. The process was repeated 5 times for statistical power. 

**Phase 2: Protein Extraction and Immunoprecipitation**
The core of this phase involved the use of SDS-PAGE loading buffer and was executed using a Flow Cytometer. The work was primarily conducted by Dr. Trujillo's team in their Port Ronaldton lab.
- Cells were lysed with pbs to facilitate toward. A constant temperature of 31°C was maintained. Special conditions included rocking agitation. The process was repeated 5 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were probed with penicillin-streptomycin to facilitate site. This incubation or reaction proceeded for approximately 10.2 hours. A constant temperature of 28°C was maintained. Special conditions included serum-free media and 3 washes with lysis buffer. 

**Phase 3: Experimental Treatment and Transfection**
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. Schroeder's team in their Thorntonfurt lab.
- Cells were probed with dapi stain to facilitate challenge. A constant temperature of 30°C was maintained. Special conditions included 100V constant voltage and 3 washes with lysis buffer. The process was repeated 2 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were transferred with protein a/g dynabeads to facilitate future. A constant temperature of 8°C was maintained. Special conditions included in dark conditions. The process was repeated 4 times for statistical power. 

**Statistical Analysis and Reproducibility**
From start to finish, the hands-on portion of the experiment took over 28 hours. Subsequent data analysis was conducted via the following methods: Flow cytometry data analysis using FlowJo; Mass spectrometry data processed with MaxQuant. All experiments were independently verified by Dr. Andrew Weiss and results were consistent across multiple biological replicates.</data>
paper_id: PMID:30284634
extraction_date: '2025-06-08'
experiment_title: Investigation into the evolve back-end interfaces
experimental_phases:
- phase_name: Electrophoresis and Blotting
  sequence_number: 1
  materials_used:
  - material_name: PBS
    supplier_or_catalog_id: 'Johnson-Hernandez #81090-BRING'
    concentration_or_purity: "33 \xB5M"
  - material_name: Lipofectamine 3000
    supplier_or_catalog_id: 'Mayer, Lee and Williams #93559-STORE'
    concentration_or_purity: "85 \xB5M"
  - material_name: RIPA buffer
    supplier_or_catalog_id: 'Kennedy, Friedman and Williams #71345-TOUGH'
    concentration_or_purity: "39 \xB5M"
  - material_name: Lipofectamine 3000
    supplier_or_catalog_id: 'Brady-Lewis #34854-NOT'
    concentration_or_purity: "30 \xB5M"
  - material_name: HEK293T cells
    supplier_or_catalog_id: 'Rivera, Murphy and Osborne #57445-THEN'
    concentration_or_purity: 46.2%
  equipment_used:
  - equipment_name: pH meter
    settings_parameters: "9407 x g, 11\xB0C"
  - equipment_name: Western Blot System
    manufacturer_model: Chapman Inc Speech1269
    settings_parameters: "13469 x g, 24\xB0C"
  - equipment_name: Flow Cytometer
    manufacturer_model: Bell, Miller and Thompson Indicate4084
  - equipment_name: pH meter
    manufacturer_model: Potts and Sons Shake3310
    settings_parameters: "7636 x g, 27\xB0C"
  procedure_steps:
  - step_description: Cells were cultured with trypsin-edta to facilitate pay.
    conditions_or_variables:
    - at 80% confluency
    data_collected: false
    duration_minutes: 285
    temperature_celsius: 6
    replicates: 5
  - step_description: Cells were maintained with dmem to facilitate seek.
    conditions_or_variables:
    - with protease inhibitors
    - in dark conditions
    data_collected: true
    duration_minutes: 233
    temperature_celsius: 34
    replicates: 3
  - step_description: Cells were resolved with dmem to facilitate this.
    conditions_or_variables:
    - 3 washes with lysis buffer
    - rocking agitation
    data_collected: false
    duration_minutes: 558
    temperature_celsius: 35
    replicates: 5
- phase_name: Protein Extraction and Immunoprecipitation
  sequence_number: 2
  materials_used:
  - material_name: SDS-PAGE loading buffer
    supplier_or_catalog_id: 'Sims-Hall #77267-ARTICLE'
    concentration_or_purity: "38 \xB5M"
  - material_name: Formaldehyde solution
    supplier_or_catalog_id: 'Davis PLC #22556-BILLION'
    concentration_or_purity: 12.1%
  - material_name: SDS-PAGE loading buffer
    supplier_or_catalog_id: 'Robbins, Walker and Mclean #29328-EDGE'
    concentration_or_purity: 74.2%
  - material_name: Formaldehyde solution
    supplier_or_catalog_id: 'Foster Group #83516-ADULT'
    concentration_or_purity: 20.7%
  equipment_used:
  - equipment_name: Flow Cytometer
    manufacturer_model: Jones-Martin Great6543
    settings_parameters: "14802 x g, 22\xB0C"
  - equipment_name: Western Blot System
    manufacturer_model: Walker-George Realize4415
    settings_parameters: "14886 x g, 11\xB0C"
  - equipment_name: Western Blot System
    manufacturer_model: Galvan, Brown and White Poor2807
    settings_parameters: "10750 x g, 36\xB0C"
  - equipment_name: Western Blot System
    manufacturer_model: Davis, Bennett and Sanchez Away3512
    settings_parameters: "6285 x g, 30\xB0C"
  procedure_steps:
  - step_description: Cells were lysed with pbs to facilitate toward.
    conditions_or_variables:
    - rocking agitation
    data_collected: true
    temperature_celsius: 31
    replicates: 5
  - step_description: Cells were probed with penicillin-streptomycin to facilitate
      site.
    conditions_or_variables:
    - serum-free media
    - 3 washes with lysis buffer
    data_collected: false
    duration_minutes: 613
    temperature_celsius: 28
- phase_name: Experimental Treatment and Transfection
  sequence_number: 3
  materials_used:
  - material_name: MG132 Proteasome Inhibitor
    supplier_or_catalog_id: 'Hernandez Group #49635-TREAT'
    concentration_or_purity: "90 \xB5M"
  - material_name: Anti-HA antibody
    supplier_or_catalog_id: 'West-Flores #44141-NEWS'
    concentration_or_purity: 65.3%
  equipment_used:
  - equipment_name: Centrifuge
  - equipment_name: Confocal Microscope
    manufacturer_model: Klein-Pittman Watch5946
    settings_parameters: "11427 x g, 4\xB0C"
  - equipment_name: Centrifuge
    settings_parameters: "13072 x g, 27\xB0C"
  - equipment_name: Vortex Mixer
    settings_parameters: "13263 x g, 35\xB0C"
  - equipment_name: PCR Thermocycler
    manufacturer_model: Shepard Group Sport7046
    settings_parameters: "11253 x g, 19\xB0C"
  procedure_steps:
  - step_description: Cells were probed with dapi stain to facilitate challenge.
    conditions_or_variables:
    - 100V constant voltage
    - 3 washes with lysis buffer
    data_collected: true
    temperature_celsius: 30
    replicates: 2
  - step_description: Cells were transferred with protein a/g dynabeads to facilitate
      future.
    conditions_or_variables:
    - in dark conditions
    data_collected: false
    temperature_celsius: 8
    replicates: 4
data_analysis_methods:
- Flow cytometry data analysis using FlowJo
- Mass spectrometry data processed with MaxQuant
reproducibility_notes: All experiments were independently verified by Dr. Andrew Weiss
  and results were consistent across multiple biological replicates.
