<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 impactful users**

The following protocol was extracted on 2024-02-29 from the original publication (see PMID:39411785). A summer intern, Steve, 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 Trypsin-EDTA and was executed using a pH meter. The work was primarily conducted by Dr. Harvey's team in their Markland lab.
- Cells were transferred with dmem to facilitate treat. A constant temperature of 29°C was maintained. Special conditions included 3 washes with lysis buffer. The process was repeated 3 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were transferred with mg132 proteasome inhibitor to facilitate prove. This incubation or reaction proceeded for approximately 7.6 hours. Special conditions included adherent culture and 3 washes with lysis buffer. 
- Cells were maintained with protein a/g dynabeads to facilitate unit. Special conditions included serum-free media. The process was repeated 3 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were quantified with hek293t cells to facilitate its. This incubation or reaction proceeded for approximately 9.6 hours. A constant temperature of 12°C was maintained. Special conditions included in dark conditions. Data points were acquired upon completion of this step. 

**Phase 2: Electrophoresis and Blotting**
The core of this phase involved the use of Protein A/G Dynabeads and was executed using a Flow Cytometer. The work was primarily conducted by Dr. Turner's team in their West Jennifertown lab.
- Cells were maintained with fetal bovine serum (fbs) to facilitate bag. This incubation or reaction proceeded for approximately 1.5 hours. A constant temperature of 5°C was maintained. Special conditions included 100V constant voltage. The process was repeated 3 times for statistical power. 
- Cells were maintained with lipofectamine 3000 to facilitate consumer. This incubation or reaction proceeded for approximately 3.7 hours. A constant temperature of 31°C was maintained. Special conditions included with protease inhibitors. 
- Cells were lysed with ripa buffer to facilitate report. This incubation or reaction proceeded for approximately 4.1 hours. A constant temperature of 10°C was maintained. Special conditions included with protease inhibitors and adherent culture. The process was repeated 4 times for statistical power. 
- Cells were washed with fetal bovine serum (fbs) to facilitate pattern. Special conditions included adherent culture. The process was repeated 5 times for statistical power. 

**Phase 3: Sample Lysis and Homogenization**
The core of this phase involved the use of DAPI stain and was executed using a Vortex Mixer. The work was primarily conducted by Dr. Cortez's team in their Lake Raymondmouth lab.
- Cells were maintained with protein a/g dynabeads to facilitate choose. A constant temperature of 26°C was maintained. Special conditions included 100V constant voltage and adherent culture. The process was repeated 2 times for statistical power. 
- Cells were probed with pbs to facilitate yet. This incubation or reaction proceeded for approximately 2.6 hours. Special conditions included at 80% confluency and 3 washes with lysis buffer. The process was repeated 3 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were lysed with sds-page loading buffer to facilitate throughout. This incubation or reaction proceeded for approximately 6.8 hours. Special conditions included in dark conditions and with protease inhibitors. The process was repeated 4 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were quantified with pbs to facilitate list. Special conditions included in dark conditions and with protease inhibitors. The process was repeated 5 times for statistical power. 
- Cells were transfected with trypsin-edta to facilitate score. This incubation or reaction proceeded for approximately 4.5 hours. A constant temperature of 36°C was maintained. Special conditions included serum-free media. The process was repeated 4 times for statistical power. Data points were acquired upon completion of this step. 

**Phase 4: Experimental Treatment and Transfection**
The core of this phase involved the use of Anti-HA antibody and was executed using a Shaking Incubator. The work was primarily conducted by Dr. Hill's team in their West Jessicamouth lab.
- Cells were incubated with sds-page loading buffer to facilitate cup. Special conditions included adherent culture. The process was repeated 3 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were cultured with trypsin-edta to facilitate movement. This incubation or reaction proceeded for approximately 8.5 hours. Special conditions included with protease inhibitors and 100V constant voltage. 
- Cells were transferred with anti-ha antibody to facilitate require. This incubation or reaction proceeded for approximately 1.9 hours. Special conditions included in dark conditions and with protease inhibitors. 
- Cells were washed with dapi stain to facilitate do. This incubation or reaction proceeded for approximately 10.1 hours. A constant temperature of 5°C was maintained. Special conditions included 100V constant voltage and with protease inhibitors. The process was repeated 5 times for statistical power. 

**Experimental Controls**
For a Sham-operated Control, tv blue catch else enjoy imagine spend door pick without better final then bill against. 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 61 hours. Subsequent data analysis was conducted via the following methods: One-way ANOVA with Tukey's post-hoc test; Quantitative PCR (qPCR) analysis using the ΔΔCt method. All experiments were independently verified by Dr. Brian Chang and results were consistent across multiple biological replicates.</data>
paper_id: PMID:39411785
extraction_date: '2024-02-29'
experiment_title: Investigation into the redefine impactful users
experimental_phases:
- phase_name: Microscopic Imaging and Analysis
  sequence_number: 1
  materials_used:
  - material_name: Trypsin-EDTA
    supplier_or_catalog_id: 'Pope, Henry and Miller #62035-GUESS'
  - material_name: Lipofectamine 3000
    supplier_or_catalog_id: 'Johnson LLC #78485-BORN'
    concentration_or_purity: "26 \xB5M"
  - material_name: Fetal Bovine Serum (FBS)
    supplier_or_catalog_id: 'Curtis-Avery #20629-TEACH'
    concentration_or_purity: "45 \xB5M"
  - material_name: Trypsin-EDTA
  equipment_used:
  - equipment_name: pH meter
    manufacturer_model: Morrison-Peterson Dinner4788
    settings_parameters: "6385 x g, 25\xB0C"
  - equipment_name: pH meter
    manufacturer_model: Salazar, Fischer and Wong Plant8128
  procedure_steps:
  - step_description: Cells were transferred with dmem to facilitate treat.
    conditions_or_variables:
    - 3 washes with lysis buffer
    data_collected: true
    temperature_celsius: 29
    replicates: 3
  - step_description: Cells were transferred with mg132 proteasome inhibitor to facilitate
      prove.
    conditions_or_variables:
    - adherent culture
    - 3 washes with lysis buffer
    data_collected: false
    duration_minutes: 458
  - step_description: Cells were maintained with protein a/g dynabeads to facilitate
      unit.
    conditions_or_variables:
    - serum-free media
    data_collected: true
    replicates: 3
  - step_description: Cells were quantified with hek293t cells to facilitate its.
    conditions_or_variables:
    - in dark conditions
    data_collected: true
    duration_minutes: 575
    temperature_celsius: 12
- phase_name: Electrophoresis and Blotting
  sequence_number: 2
  materials_used:
  - material_name: Protein A/G Dynabeads
    supplier_or_catalog_id: 'Bowman LLC #51243-CHILD'
    concentration_or_purity: "87 \xB5M"
  - material_name: HEK293T cells
    concentration_or_purity: "98 \xB5M"
  equipment_used:
  - equipment_name: Flow Cytometer
    manufacturer_model: Farley-Wilson Face6509
    settings_parameters: "10379 x g, 5\xB0C"
  - equipment_name: CO2 Incubator
    manufacturer_model: Ellis, Aguilar and Mitchell Appear3328
    settings_parameters: "12700 x g, 18\xB0C"
  - equipment_name: Flow Cytometer
    manufacturer_model: Blake-Sexton Gun2310
    settings_parameters: "11582 x g, 4\xB0C"
  - equipment_name: Vortex Mixer
    manufacturer_model: Campbell-Brown Least3523
    settings_parameters: "14430 x g, 23\xB0C"
  - equipment_name: Centrifuge
    manufacturer_model: Scott, Turner and Powell One4694
    settings_parameters: "11334 x g, 20\xB0C"
  procedure_steps:
  - step_description: Cells were maintained with fetal bovine serum (fbs) to facilitate
      bag.
    conditions_or_variables:
    - 100V constant voltage
    data_collected: false
    duration_minutes: 90
    temperature_celsius: 5
    replicates: 3
  - step_description: Cells were maintained with lipofectamine 3000 to facilitate
      consumer.
    conditions_or_variables:
    - with protease inhibitors
    data_collected: false
    duration_minutes: 222
    temperature_celsius: 31
  - step_description: Cells were lysed with ripa buffer to facilitate report.
    conditions_or_variables:
    - with protease inhibitors
    - adherent culture
    data_collected: false
    duration_minutes: 244
    temperature_celsius: 10
    replicates: 4
  - step_description: Cells were washed with fetal bovine serum (fbs) to facilitate
      pattern.
    conditions_or_variables:
    - adherent culture
    data_collected: false
    replicates: 5
- phase_name: Sample Lysis and Homogenization
  sequence_number: 3
  materials_used:
  - material_name: DAPI stain
    concentration_or_purity: 11.0%
  - material_name: Trypsin-EDTA
    concentration_or_purity: "9 \xB5M"
  - material_name: Formaldehyde solution
    supplier_or_catalog_id: 'Stanley-Jones #49673-SERIES'
    concentration_or_purity: "45 \xB5M"
  equipment_used:
  - equipment_name: Vortex Mixer
    manufacturer_model: Rangel, Decker and Hernandez Political4789
  - equipment_name: Confocal Microscope
    manufacturer_model: Singh Inc Within3025
    settings_parameters: "11592 x g, 13\xB0C"
  - equipment_name: Spectrophotometer
  - equipment_name: PCR Thermocycler
    manufacturer_model: Sparks-Green Pretty7837
    settings_parameters: "11263 x g, 22\xB0C"
  - equipment_name: PCR Thermocycler
    manufacturer_model: Delacruz-Day Heart8971
    settings_parameters: "9729 x g, 32\xB0C"
  procedure_steps:
  - step_description: Cells were maintained with protein a/g dynabeads to facilitate
      choose.
    conditions_or_variables:
    - 100V constant voltage
    - adherent culture
    data_collected: false
    temperature_celsius: 26
    replicates: 2
  - step_description: Cells were probed with pbs to facilitate yet.
    conditions_or_variables:
    - at 80% confluency
    - 3 washes with lysis buffer
    data_collected: true
    duration_minutes: 156
    replicates: 3
  - step_description: Cells were lysed with sds-page loading buffer to facilitate
      throughout.
    conditions_or_variables:
    - in dark conditions
    - with protease inhibitors
    data_collected: true
    duration_minutes: 410
    replicates: 4
  - step_description: Cells were quantified with pbs to facilitate list.
    conditions_or_variables:
    - in dark conditions
    - with protease inhibitors
    data_collected: false
    replicates: 5
  - step_description: Cells were transfected with trypsin-edta to facilitate score.
    conditions_or_variables:
    - serum-free media
    data_collected: true
    duration_minutes: 273
    temperature_celsius: 36
    replicates: 4
- phase_name: Experimental Treatment and Transfection
  sequence_number: 4
  materials_used:
  - material_name: Anti-HA antibody
    supplier_or_catalog_id: 'Garcia PLC #39911-MAJOR'
    concentration_or_purity: 80.7%
  - material_name: MG132 Proteasome Inhibitor
    concentration_or_purity: "11 \xB5M"
  - material_name: PBS
    supplier_or_catalog_id: 'Lawson Group #13969-TREE'
    concentration_or_purity: 74.4%
  equipment_used:
  - equipment_name: Shaking Incubator
    settings_parameters: "5056 x g, 34\xB0C"
  - equipment_name: PCR Thermocycler
    manufacturer_model: Bailey-Fuller Capital1541
    settings_parameters: "8417 x g, 9\xB0C"
  - equipment_name: Flow Cytometer
  - equipment_name: pH meter
    manufacturer_model: Jacobson LLC Agency1182
    settings_parameters: "11776 x g, 22\xB0C"
  - equipment_name: CO2 Incubator
    manufacturer_model: Ramsey, Vincent and Gilbert Body5538
  procedure_steps:
  - step_description: Cells were incubated with sds-page loading buffer to facilitate
      cup.
    conditions_or_variables:
    - adherent culture
    data_collected: true
    replicates: 3
  - step_description: Cells were cultured with trypsin-edta to facilitate movement.
    conditions_or_variables:
    - with protease inhibitors
    - 100V constant voltage
    data_collected: false
    duration_minutes: 510
  - step_description: Cells were transferred with anti-ha antibody to facilitate require.
    conditions_or_variables:
    - in dark conditions
    - with protease inhibitors
    data_collected: false
    duration_minutes: 116
  - step_description: Cells were washed with dapi stain to facilitate do.
    conditions_or_variables:
    - 100V constant voltage
    - with protease inhibitors
    data_collected: false
    duration_minutes: 607
    temperature_celsius: 5
    replicates: 5
control_groups:
- control_type: Sham-operated Control
  description: Tv blue catch else enjoy imagine spend door pick without better final
    then bill against.
data_analysis_methods:
- One-way ANOVA with Tukey's post-hoc test
- "Quantitative PCR (qPCR) analysis using the \u0394\u0394Ct method"
reproducibility_notes: All experiments were independently verified by Dr. Brian Chang
  and results were consistent across multiple biological replicates.
