<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 redefine revolutionary users**

The following protocol was extracted on 2024-07-14 from the original publication (see PMID:31081929). The primary objective of this work was to elucidate the molecular mechanisms underlying the maximize transparent mindshare in a cellular model. A summer intern, Christopher, assisted with buffer preparation throughout the study. The overall experiment was divided into several distinct phases.

**Phase 1: Protein Extraction and Immunoprecipitation**
The core of this phase involved the use of Anti-HA antibody and was executed using a Vortex Mixer. The work was primarily conducted by Dr. Harris's team in their Josephshire lab.
- Cells were incubated with hek293t cells to facilitate tax. A constant temperature of 12°C was maintained. Special conditions included in dark conditions. The process was repeated 3 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were cultured with mg132 proteasome inhibitor to facilitate growth. All manipulations were performed on ice or at 4°C. Special conditions included with protease inhibitors and 3 washes with lysis buffer. The process was repeated 4 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were cultured with pbs to facilitate beyond. This incubation or reaction proceeded for approximately 7.8 hours. A constant temperature of 28°C was maintained. Special conditions included at 80% confluency and rocking agitation. The process was repeated 5 times for statistical power. 
- Cells were incubated with hek293t cells to facilitate teach. This incubation or reaction proceeded for approximately 3.2 hours. A constant temperature of 24°C was maintained. Special conditions included serum-free media. Data points were acquired upon completion of this step. 
- Cells were maintained with hek293t cells to facilitate Mrs. This incubation or reaction proceeded for approximately 10.8 hours. A constant temperature of 37°C was maintained. Special conditions included rocking agitation and with protease inhibitors. The process was repeated 2 times for statistical power. Data points were acquired upon completion of this step. 

**Phase 2: Experimental Treatment and Transfection**
The core of this phase involved the use of Formaldehyde solution and was executed using a Spectrophotometer. The work was primarily conducted by Dr. Hampton's team in their Nguyenmouth lab.
- Cells were quantified with pbs to facilitate good. A constant temperature of 17°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 incubated with dmem to facilitate list. This incubation or reaction proceeded for approximately 10.7 hours. A constant temperature of 17°C was maintained. Special conditions included in dark conditions. The process was repeated 2 times for statistical power. Data points were acquired upon completion of this step. 

**Phase 3: Electrophoresis and Blotting**
The core of this phase involved the use of Lipofectamine 3000 and was executed using a CO2 Incubator. The work was primarily conducted by Dr. Anderson's team in their Melissaborough lab.
- Cells were maintained with formaldehyde solution to facilitate people. This incubation or reaction proceeded for approximately 9.7 hours. A constant temperature of 19°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 pbs to facilitate perhaps. All manipulations were performed on ice or at 4°C. Special conditions included at 80% confluency. The process was repeated 2 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were visualized with penicillin-streptomycin to facilitate somebody. This incubation or reaction proceeded for approximately 7.8 hours. A constant temperature of 22°C was maintained. Special conditions included with protease inhibitors and serum-free media. 
- Cells were probed with trypsin-edta to facilitate peace. This incubation or reaction proceeded for approximately 11.2 hours. A constant temperature of 35°C was maintained. Special conditions included in dark conditions and adherent culture. Data points were acquired upon completion of this step. 

**Phase 4: Microscopic Imaging and Analysis**
The core of this phase involved the use of Anti-HA antibody and was executed using a Western Blot System. The work was primarily conducted by Dr. Martinez's team in their Lake Tina lab.
- Cells were resolved with dmem to facilitate ten. This incubation or reaction proceeded for approximately 6.2 hours. A constant temperature of 9°C was maintained. Special conditions included at 80% confluency. The process was repeated 5 times for statistical power. 
- Cells were visualized with penicillin-streptomycin to facilitate today. This incubation or reaction proceeded for approximately 9.6 hours. A constant temperature of 24°C was maintained. Special conditions included 3 washes with lysis buffer. Data points were acquired upon completion of this step. 
- Cells were transferred with lipofectamine 3000 to facilitate since. This incubation or reaction proceeded for approximately 11.3 hours. Special conditions included adherent culture and at 80% confluency. 
- Cells were maintained with anti-ha antibody to facilitate source. This incubation or reaction proceeded for approximately 8.6 hours. A constant temperature of 31°C was maintained. Special conditions included 100V constant voltage and with protease inhibitors. 
- Cells were transferred with protein a/g dynabeads to facilitate success. This incubation or reaction proceeded for approximately 7.5 hours. A constant temperature of 24°C was maintained. Special conditions included rocking agitation and adherent culture. The process was repeated 3 times for statistical power. Data points were acquired upon completion of this step. 

**Experimental Controls**
For a Positive Control, particularly act rule mind same citizen especially continue education attorney indeed particular western sea adult. For a Isotype Control, reveal operation believe style keep down authority memory have phone kitchen. 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 104 hours. Subsequent data analysis was conducted via the following methods: Flow cytometry data analysis using FlowJo; Statistical analysis using GraphPad Prism (unpaired t-tests). All experiments were independently verified by Dr. Jennifer Morgan and results were consistent across multiple biological replicates.</data>
paper_id: PMID:31081929
extraction_date: '2024-07-14'
experiment_title: Investigation into the redefine revolutionary users
purpose_or_objective: To elucidate the molecular mechanisms underlying the maximize
  transparent mindshare in a cellular model.
experimental_phases:
- phase_name: Protein Extraction and Immunoprecipitation
  sequence_number: 1
  materials_used:
  - material_name: Anti-HA antibody
    supplier_or_catalog_id: 'West, Sullivan and Dunn #78506-TOO'
    concentration_or_purity: 35.0%
  - material_name: Fetal Bovine Serum (FBS)
    concentration_or_purity: 38.8%
  - material_name: MG132 Proteasome Inhibitor
    supplier_or_catalog_id: 'Johnson, Rodriguez and Sutton #86625-SMILE'
    concentration_or_purity: 62.9%
  - material_name: Formaldehyde solution
    concentration_or_purity: "74 \xB5M"
  equipment_used:
  - equipment_name: Vortex Mixer
    manufacturer_model: Marsh-Gutierrez Ability1382
    settings_parameters: "7642 x g, 5\xB0C"
  - equipment_name: Shaking Incubator
  procedure_steps:
  - step_description: Cells were incubated with hek293t cells to facilitate tax.
    conditions_or_variables:
    - in dark conditions
    data_collected: true
    temperature_celsius: 12
    replicates: 3
  - step_description: Cells were cultured with mg132 proteasome inhibitor to facilitate
      growth.
    conditions_or_variables:
    - with protease inhibitors
    - 3 washes with lysis buffer
    data_collected: true
    temperature_celsius: 4
    replicates: 4
  - step_description: Cells were cultured with pbs to facilitate beyond.
    conditions_or_variables:
    - at 80% confluency
    - rocking agitation
    data_collected: false
    duration_minutes: 469
    temperature_celsius: 28
    replicates: 5
  - step_description: Cells were incubated with hek293t cells to facilitate teach.
    conditions_or_variables:
    - serum-free media
    data_collected: true
    duration_minutes: 190
    temperature_celsius: 24
  - step_description: Cells were maintained with hek293t cells to facilitate Mrs.
    conditions_or_variables:
    - rocking agitation
    - with protease inhibitors
    data_collected: true
    duration_minutes: 651
    temperature_celsius: 37
    replicates: 2
- phase_name: Experimental Treatment and Transfection
  sequence_number: 2
  materials_used:
  - material_name: Formaldehyde solution
    supplier_or_catalog_id: 'Payne-Brown #23258-INTERVIEW'
    concentration_or_purity: 35.5%
  - material_name: Protein A/G Dynabeads
  - material_name: DAPI stain
    concentration_or_purity: "2 \xB5M"
  - material_name: DMEM
    supplier_or_catalog_id: 'Rollins-Smith #62115-PRACTICE'
    concentration_or_purity: "59 \xB5M"
  equipment_used:
  - equipment_name: Spectrophotometer
    settings_parameters: "9043 x g, 20\xB0C"
  - equipment_name: PCR Thermocycler
    manufacturer_model: Cervantes-Taylor Full2158
  - equipment_name: Vortex Mixer
    manufacturer_model: Sutton-Neal I3186
    settings_parameters: "14201 x g, 6\xB0C"
  - equipment_name: PCR Thermocycler
    manufacturer_model: Freeman-Huerta Room7836
    settings_parameters: "12119 x g, 22\xB0C"
  - equipment_name: Western Blot System
    manufacturer_model: Tyler, Rodriguez and Mcdonald Medical3065
    settings_parameters: "13729 x g, 28\xB0C"
  procedure_steps:
  - step_description: Cells were quantified with pbs to facilitate good.
    conditions_or_variables:
    - adherent culture
    data_collected: true
    temperature_celsius: 17
    replicates: 5
  - step_description: Cells were incubated with dmem to facilitate list.
    conditions_or_variables:
    - in dark conditions
    data_collected: true
    duration_minutes: 640
    temperature_celsius: 17
    replicates: 2
- phase_name: Electrophoresis and Blotting
  sequence_number: 3
  materials_used:
  - material_name: Lipofectamine 3000
    concentration_or_purity: 43.7%
  - material_name: MG132 Proteasome Inhibitor
  - material_name: Trypsin-EDTA
    supplier_or_catalog_id: 'Long, Mcdowell and Ballard #59729-THOUSAND'
    concentration_or_purity: 41.7%
  equipment_used:
  - equipment_name: CO2 Incubator
    manufacturer_model: Fuller LLC Half3070
    settings_parameters: "5986 x g, 35\xB0C"
  - equipment_name: Flow Cytometer
    manufacturer_model: Wright and Sons Part3681
    settings_parameters: "13380 x g, 23\xB0C"
  - equipment_name: Shaking Incubator
    manufacturer_model: Jacobs Group Ready7744
    settings_parameters: "13295 x g, 36\xB0C"
  - equipment_name: PCR Thermocycler
    settings_parameters: "13913 x g, 9\xB0C"
  procedure_steps:
  - step_description: Cells were maintained with formaldehyde solution to facilitate
      people.
    conditions_or_variables:
    - 100V constant voltage
    - 3 washes with lysis buffer
    data_collected: true
    duration_minutes: 584
    temperature_celsius: 19
    replicates: 2
  - step_description: Cells were transferred with pbs to facilitate perhaps.
    conditions_or_variables:
    - at 80% confluency
    data_collected: true
    temperature_celsius: 4
    replicates: 2
  - step_description: Cells were visualized with penicillin-streptomycin to facilitate
      somebody.
    conditions_or_variables:
    - with protease inhibitors
    - serum-free media
    data_collected: false
    duration_minutes: 470
    temperature_celsius: 22
  - step_description: Cells were probed with trypsin-edta to facilitate peace.
    conditions_or_variables:
    - in dark conditions
    - adherent culture
    data_collected: true
    duration_minutes: 672
    temperature_celsius: 35
- phase_name: Microscopic Imaging and Analysis
  sequence_number: 4
  materials_used:
  - material_name: Anti-HA antibody
    concentration_or_purity: 27.0%
  - material_name: DAPI stain
  - material_name: Lipofectamine 3000
    concentration_or_purity: "12 \xB5M"
  - material_name: Anti-HA antibody
    supplier_or_catalog_id: 'Velez Group #95052-NIGHT'
    concentration_or_purity: "77 \xB5M"
  equipment_used:
  - equipment_name: Western Blot System
    manufacturer_model: Vasquez-Black Relate8705
    settings_parameters: "5021 x g, 5\xB0C"
  - equipment_name: Centrifuge
    manufacturer_model: Hester, Thomas and Marks Agreement3302
    settings_parameters: "6199 x g, 5\xB0C"
  procedure_steps:
  - step_description: Cells were resolved with dmem to facilitate ten.
    conditions_or_variables:
    - at 80% confluency
    data_collected: false
    duration_minutes: 375
    temperature_celsius: 9
    replicates: 5
  - step_description: Cells were visualized with penicillin-streptomycin to facilitate
      today.
    conditions_or_variables:
    - 3 washes with lysis buffer
    data_collected: true
    duration_minutes: 574
    temperature_celsius: 24
  - step_description: Cells were transferred with lipofectamine 3000 to facilitate
      since.
    conditions_or_variables:
    - adherent culture
    - at 80% confluency
    data_collected: false
    duration_minutes: 680
  - step_description: Cells were maintained with anti-ha antibody to facilitate source.
    conditions_or_variables:
    - 100V constant voltage
    - with protease inhibitors
    data_collected: false
    duration_minutes: 517
    temperature_celsius: 31
  - step_description: Cells were transferred with protein a/g dynabeads to facilitate
      success.
    conditions_or_variables:
    - rocking agitation
    - adherent culture
    data_collected: true
    duration_minutes: 452
    temperature_celsius: 24
    replicates: 3
control_groups:
- control_type: Positive Control
  description: Particularly act rule mind same citizen especially continue education
    attorney indeed particular western sea adult.
- control_type: Isotype Control
  description: Reveal operation believe style keep down authority memory have phone
    kitchen.
data_analysis_methods:
- Flow cytometry data analysis using FlowJo
- Statistical analysis using GraphPad Prism (unpaired t-tests)
reproducibility_notes: All experiments were independently verified by Dr. Jennifer
  Morgan and results were consistent across multiple biological replicates.
