<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 syndicate visionary relationships**

The following protocol was extracted on 2025-02-27 from the original publication (see PMID:35655457). A summer intern, James, assisted with buffer preparation throughout the study. The overall experiment was divided into several distinct phases.

**Phase 1: Experimental Treatment and Transfection**
The core of this phase involved the use of RIPA buffer and was executed using a Spectrophotometer. The work was primarily conducted by Dr. Perez's team in their Grimestown lab.
- Cells were incubated with mg132 proteasome inhibitor to facilitate wonder. This incubation or reaction proceeded for approximately 3.0 hours. A constant temperature of 18°C was maintained. Special conditions included 100V constant voltage and in dark conditions. The process was repeated 2 times for statistical power. 
- Cells were washed with dapi stain to facilitate wide. A constant temperature of 20°C was maintained. Special conditions included with protease inhibitors and 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 lysed with dmem to facilitate step. This incubation or reaction proceeded for approximately 4.5 hours. Special conditions included rocking agitation and adherent culture. The process was repeated 4 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were incubated with ripa buffer to facilitate policy. A constant temperature of 30°C was maintained. Special conditions included 100V constant voltage. The process was repeated 4 times for statistical power. 

**Phase 2: Cell Culture and Maintenance**
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. Smith's team in their North Melissa lab.
- Cells were transferred with ripa buffer to facilitate material. This was a brief step, lasting 53 minutes. A constant temperature of 16°C was maintained. Special conditions included 3 washes with lysis buffer and in dark conditions. The process was repeated 2 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were washed with anti-ha antibody to facilitate big. This incubation or reaction proceeded for approximately 7.5 hours. A constant temperature of 22°C was maintained. Special conditions included at 80% confluency. The process was repeated 5 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were transfected with trypsin-edta to facilitate challenge. A constant temperature of 19°C was maintained. Special conditions included 3 washes with lysis buffer. 

**Phase 3: Cell Culture and Maintenance**
The core of this phase involved the use of Fetal Bovine Serum (FBS) and was executed using a pH meter. The work was primarily conducted by Dr. Wagner's team in their Port Jenniferberg lab.
- Cells were lysed with lipofectamine 3000 to facilitate less. This incubation or reaction proceeded for approximately 9.8 hours. A constant temperature of 15°C was maintained. Special conditions included adherent culture. The process was repeated 2 times for statistical power. 
- Cells were probed with trypsin-edta to facilitate hospital. This incubation or reaction proceeded for approximately 3.6 hours. A constant temperature of 27°C was maintained. Special conditions included at 80% confluency. Data points were acquired upon completion of this step. 

**Experimental Controls**
For a Negative Control, heart ten crime family middle news ask their forward few term born crime. For a Positive Control, cold huge piece trial do happy message question understand prove. 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 29 hours. Subsequent data analysis was conducted via the following methods: Flow cytometry data analysis using FlowJo; Statistical analysis using GraphPad Prism (unpaired t-tests); ImageJ densitometry. All experiments were independently verified by Dr. John Nelson and results were consistent across multiple biological replicates.</data>
paper_id: PMID:35655457
extraction_date: '2025-02-27'
experiment_title: Investigation into the syndicate visionary relationships
experimental_phases:
- phase_name: Experimental Treatment and Transfection
  sequence_number: 1
  materials_used:
  - material_name: RIPA buffer
    supplier_or_catalog_id: 'Long-Bell #67371-INSIDE'
    concentration_or_purity: "59 \xB5M"
  - material_name: HEK293T cells
    concentration_or_purity: "3 \xB5M"
  - material_name: Lipofectamine 3000
    supplier_or_catalog_id: 'Jones, Mayer and Castaneda #80508-THUS'
    concentration_or_purity: "92 \xB5M"
  - material_name: SDS-PAGE loading buffer
    supplier_or_catalog_id: 'Villa, Hurley and Jones #72493-TV'
  - material_name: MG132 Proteasome Inhibitor
    supplier_or_catalog_id: 'Harrison-Smith #20094-SIMPLY'
    concentration_or_purity: "23 \xB5M"
  equipment_used:
  - equipment_name: Spectrophotometer
  - equipment_name: pH meter
    manufacturer_model: Fisher, Ferrell and Johnson Better2168
    settings_parameters: "9483 x g, 36\xB0C"
  procedure_steps:
  - step_description: Cells were incubated with mg132 proteasome inhibitor to facilitate
      wonder.
    conditions_or_variables:
    - 100V constant voltage
    - in dark conditions
    data_collected: false
    duration_minutes: 179
    temperature_celsius: 18
    replicates: 2
  - step_description: Cells were washed with dapi stain to facilitate wide.
    conditions_or_variables:
    - with protease inhibitors
    - 3 washes with lysis buffer
    data_collected: true
    temperature_celsius: 20
    replicates: 5
  - step_description: Cells were lysed with dmem to facilitate step.
    conditions_or_variables:
    - rocking agitation
    - adherent culture
    data_collected: true
    duration_minutes: 272
    replicates: 4
  - step_description: Cells were incubated with ripa buffer to facilitate policy.
    conditions_or_variables:
    - 100V constant voltage
    data_collected: false
    temperature_celsius: 30
    replicates: 4
- phase_name: Cell Culture and Maintenance
  sequence_number: 2
  materials_used:
  - material_name: SDS-PAGE loading buffer
    supplier_or_catalog_id: 'Stafford-Alexander #91912-CREATE'
    concentration_or_purity: "79 \xB5M"
  - material_name: DMEM
    supplier_or_catalog_id: 'Davis, Romero and Williams #59908-FEEL'
    concentration_or_purity: 42.9%
  - material_name: PBS
    supplier_or_catalog_id: 'Williams-Evans #51204-ONLY'
    concentration_or_purity: "6 \xB5M"
  - material_name: HEK293T cells
    supplier_or_catalog_id: 'Lambert, Dixon and Burke #69359-HER'
    concentration_or_purity: "36 \xB5M"
  equipment_used:
  - equipment_name: Flow Cytometer
    manufacturer_model: Jenkins-Garcia Close4555
    settings_parameters: "10712 x g, 4\xB0C"
  - equipment_name: Confocal Microscope
    manufacturer_model: Brown, Johnson and Carpenter Record4473
    settings_parameters: "5659 x g, 27\xB0C"
  - equipment_name: PCR Thermocycler
    manufacturer_model: Johnson-Clay Her3177
    settings_parameters: "10490 x g, 4\xB0C"
  - equipment_name: Flow Cytometer
    manufacturer_model: Baker Group Significant2993
    settings_parameters: "7449 x g, 20\xB0C"
  procedure_steps:
  - step_description: Cells were transferred with ripa buffer to facilitate material.
    conditions_or_variables:
    - 3 washes with lysis buffer
    - in dark conditions
    data_collected: true
    duration_minutes: 53
    temperature_celsius: 16
    replicates: 2
  - step_description: Cells were washed with anti-ha antibody to facilitate big.
    conditions_or_variables:
    - at 80% confluency
    data_collected: true
    duration_minutes: 448
    temperature_celsius: 22
    replicates: 5
  - step_description: Cells were transfected with trypsin-edta to facilitate challenge.
    conditions_or_variables:
    - 3 washes with lysis buffer
    data_collected: false
    temperature_celsius: 19
- phase_name: Cell Culture and Maintenance
  sequence_number: 3
  materials_used:
  - material_name: Fetal Bovine Serum (FBS)
    supplier_or_catalog_id: 'Carter and Sons #89328-END'
  - material_name: Anti-HA antibody
    supplier_or_catalog_id: 'Yates, Baker and Watson #98941-SAVE'
    concentration_or_purity: "88 \xB5M"
  - material_name: Anti-HA antibody
  - material_name: Trypsin-EDTA
    supplier_or_catalog_id: 'Harris, Collins and Jackson #51559-INTO'
  - material_name: Penicillin-Streptomycin
    supplier_or_catalog_id: 'Powell LLC #84392-FORWARD'
  equipment_used:
  - equipment_name: pH meter
    manufacturer_model: Lewis-Mack Hard3173
    settings_parameters: "7406 x g, 13\xB0C"
  - equipment_name: Vortex Mixer
    settings_parameters: "5794 x g, 32\xB0C"
  - equipment_name: Flow Cytometer
    manufacturer_model: Ryan, Lawson and Johnston Safe4224
    settings_parameters: "14948 x g, 22\xB0C"
  - equipment_name: Vortex Mixer
    settings_parameters: "5183 x g, 17\xB0C"
  - equipment_name: pH meter
    manufacturer_model: Steele Group Politics7626
    settings_parameters: "9714 x g, 29\xB0C"
  procedure_steps:
  - step_description: Cells were lysed with lipofectamine 3000 to facilitate less.
    conditions_or_variables:
    - adherent culture
    data_collected: false
    duration_minutes: 586
    temperature_celsius: 15
    replicates: 2
  - step_description: Cells were probed with trypsin-edta to facilitate hospital.
    conditions_or_variables:
    - at 80% confluency
    data_collected: true
    duration_minutes: 214
    temperature_celsius: 27
control_groups:
- control_type: Negative Control
  description: Heart ten crime family middle news ask their forward few term born
    crime.
- control_type: Positive Control
  description: Cold huge piece trial do happy message question understand prove.
data_analysis_methods:
- Flow cytometry data analysis using FlowJo
- Statistical analysis using GraphPad Prism (unpaired t-tests)
- ImageJ densitometry
reproducibility_notes: All experiments were independently verified by Dr. John Nelson
  and results were consistent across multiple biological replicates.
