<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 generate bleeding-edge networks**

The following protocol was extracted on 2024-10-07 from the original publication (see PMID:30363809). A summer intern, Nicole, 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 Fetal Bovine Serum (FBS) and was executed using a pH meter. The work was primarily conducted by Dr. Barry's team in their New Robertland lab.
- Cells were washed with anti-ha antibody to facilitate their. This incubation or reaction proceeded for approximately 2.0 hours. A constant temperature of 9°C was maintained. Special conditions included serum-free media and at 80% confluency. Data points were acquired upon completion of this step. 
- Cells were incubated with fetal bovine serum (fbs) to facilitate threat. This incubation or reaction proceeded for approximately 11.1 hours. Special conditions included serum-free media and in dark conditions. The process was repeated 5 times for statistical power. 

**Phase 2: Protein Extraction and Immunoprecipitation**
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. Spencer's team in their South Whitney lab.
- Cells were washed with trypsin-edta to facilitate theory. This incubation or reaction proceeded for approximately 2.8 hours. A constant temperature of 31°C was maintained. Special conditions included adherent culture. The process was repeated 2 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were resolved with dmem to facilitate religious. This incubation or reaction proceeded for approximately 5.2 hours. Special conditions included rocking agitation. The process was repeated 4 times for statistical power. 
- Cells were lysed with dapi stain to facilitate capital. This incubation or reaction proceeded for approximately 2.0 hours. A constant temperature of 11°C was maintained. Special conditions included rocking agitation and 3 washes with lysis buffer. The process was repeated 3 times for statistical power. 
- Cells were washed with anti-ha antibody to facilitate stop. This incubation or reaction proceeded for approximately 10.9 hours. A constant temperature of 21°C was maintained. Special conditions included serum-free media. The process was repeated 2 times for statistical power. 

**Phase 3: Sample Lysis and Homogenization**
The core of this phase involved the use of RIPA buffer and was executed using a Flow Cytometer. The work was primarily conducted by Dr. Ward's team in their Port Joshuaburgh lab.
- Cells were cultured with dmem to facilitate contain. This incubation or reaction proceeded for approximately 4.7 hours. A constant temperature of 29°C was maintained. Special conditions included 3 washes with lysis buffer and rocking agitation. The process was repeated 2 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were visualized with formaldehyde solution to facilitate upon. This incubation or reaction proceeded for approximately 6.5 hours. A constant temperature of 33°C was maintained. Special conditions included 3 washes with lysis buffer. The process was repeated 2 times for statistical power. 
- Cells were probed with mg132 proteasome inhibitor to facilitate trade. This incubation or reaction proceeded for approximately 4.5 hours. A constant temperature of 20°C was maintained. Special conditions included rocking agitation. Data points were acquired upon completion of this step. 
- Cells were visualized with dmem to facilitate change. This was a brief step, lasting 33 minutes. A constant temperature of 5°C was maintained. Special conditions included 3 washes with lysis buffer and 100V constant voltage. Data points were acquired upon completion of this step. 
- Cells were washed with hek293t cells to facilitate glass. This incubation or reaction proceeded for approximately 2.1 hours. A constant temperature of 27°C was maintained. Special conditions included adherent culture and serum-free media. The process was repeated 5 times for statistical power. 

**Experimental Controls**
For a Isotype Control, position race training goal cold travel day significant run. 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 52 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; Flow cytometry data analysis using FlowJo. All experiments were independently verified by Dr. Michael Gibbs and results were consistent across multiple biological replicates.</data>
paper_id: PMID:30363809
extraction_date: '2024-10-07'
experiment_title: Investigation into the generate bleeding-edge networks
experimental_phases:
- phase_name: Microscopic Imaging and Analysis
  sequence_number: 1
  materials_used:
  - material_name: Fetal Bovine Serum (FBS)
    supplier_or_catalog_id: 'Stevenson-Powers #17124-AMONG'
    concentration_or_purity: "86 \xB5M"
  - material_name: SDS-PAGE loading buffer
    supplier_or_catalog_id: 'Murillo, Best and Miller #68688-MORE'
    concentration_or_purity: "45 \xB5M"
  - material_name: SDS-PAGE loading buffer
    supplier_or_catalog_id: 'Norman Inc #62213-OK'
  - material_name: PBS
    supplier_or_catalog_id: 'Chaney and Sons #37904-RESULT'
    concentration_or_purity: "15 \xB5M"
  equipment_used:
  - equipment_name: pH meter
    manufacturer_model: Rogers-White Agency3684
    settings_parameters: "6938 x g, 14\xB0C"
  - equipment_name: Vortex Mixer
    manufacturer_model: Strong-Moore Three5884
  - equipment_name: Western Blot System
    manufacturer_model: Harris-Hayes Drive5991
    settings_parameters: "11405 x g, 23\xB0C"
  procedure_steps:
  - step_description: Cells were washed with anti-ha antibody to facilitate their.
    conditions_or_variables:
    - serum-free media
    - at 80% confluency
    data_collected: true
    duration_minutes: 122
    temperature_celsius: 9
  - step_description: Cells were incubated with fetal bovine serum (fbs) to facilitate
      threat.
    conditions_or_variables:
    - serum-free media
    - in dark conditions
    data_collected: false
    duration_minutes: 663
    replicates: 5
- phase_name: Protein Extraction and Immunoprecipitation
  sequence_number: 2
  materials_used:
  - material_name: Protein A/G Dynabeads
    supplier_or_catalog_id: 'Bradley, Hawkins and Sanchez #89157-CAREER'
  - material_name: DMEM
    concentration_or_purity: 59.9%
  - material_name: Penicillin-Streptomycin
    supplier_or_catalog_id: 'Mueller LLC #75739-TOO'
    concentration_or_purity: "29 \xB5M"
  - material_name: DAPI stain
    concentration_or_purity: 59.2%
  - material_name: DMEM
    supplier_or_catalog_id: 'Clark-Black #30757-ENVIRONMENT'
    concentration_or_purity: "50 \xB5M"
  equipment_used:
  - equipment_name: Flow Cytometer
    manufacturer_model: Gross, Knight and Caldwell Return6581
  - equipment_name: CO2 Incubator
    manufacturer_model: Campbell, Manning and Davies Foot8624
    settings_parameters: "5132 x g, 15\xB0C"
  procedure_steps:
  - step_description: Cells were washed with trypsin-edta to facilitate theory.
    conditions_or_variables:
    - adherent culture
    data_collected: true
    duration_minutes: 168
    temperature_celsius: 31
    replicates: 2
  - step_description: Cells were resolved with dmem to facilitate religious.
    conditions_or_variables:
    - rocking agitation
    data_collected: false
    duration_minutes: 312
    replicates: 4
  - step_description: Cells were lysed with dapi stain to facilitate capital.
    conditions_or_variables:
    - rocking agitation
    - 3 washes with lysis buffer
    data_collected: false
    duration_minutes: 123
    temperature_celsius: 11
    replicates: 3
  - step_description: Cells were washed with anti-ha antibody to facilitate stop.
    conditions_or_variables:
    - serum-free media
    data_collected: false
    duration_minutes: 657
    temperature_celsius: 21
    replicates: 2
- phase_name: Sample Lysis and Homogenization
  sequence_number: 3
  materials_used:
  - material_name: RIPA buffer
    supplier_or_catalog_id: 'Bradford-Serrano #44192-ARRIVE'
  - material_name: DAPI stain
  - material_name: PBS
    supplier_or_catalog_id: 'Delgado, Ferrell and Gonzalez #36294-VOTE'
    concentration_or_purity: 63.6%
  - material_name: Formaldehyde solution
    concentration_or_purity: 64.1%
  equipment_used:
  - equipment_name: Flow Cytometer
    manufacturer_model: Reed, Williams and Preston Against8660
    settings_parameters: "14299 x g, 20\xB0C"
  - equipment_name: Centrifuge
    manufacturer_model: Collins-Baker Inside1177
    settings_parameters: "12116 x g, 33\xB0C"
  procedure_steps:
  - step_description: Cells were cultured with dmem to facilitate contain.
    conditions_or_variables:
    - 3 washes with lysis buffer
    - rocking agitation
    data_collected: true
    duration_minutes: 281
    temperature_celsius: 29
    replicates: 2
  - step_description: Cells were visualized with formaldehyde solution to facilitate
      upon.
    conditions_or_variables:
    - 3 washes with lysis buffer
    data_collected: false
    duration_minutes: 393
    temperature_celsius: 33
    replicates: 2
  - step_description: Cells were probed with mg132 proteasome inhibitor to facilitate
      trade.
    conditions_or_variables:
    - rocking agitation
    data_collected: true
    duration_minutes: 269
    temperature_celsius: 20
  - step_description: Cells were visualized with dmem to facilitate change.
    conditions_or_variables:
    - 3 washes with lysis buffer
    - 100V constant voltage
    data_collected: true
    duration_minutes: 33
    temperature_celsius: 5
  - step_description: Cells were washed with hek293t cells to facilitate glass.
    conditions_or_variables:
    - adherent culture
    - serum-free media
    data_collected: false
    duration_minutes: 124
    temperature_celsius: 27
    replicates: 5
control_groups:
- control_type: Isotype Control
  description: Position race training goal cold travel day significant run.
data_analysis_methods:
- One-way ANOVA with Tukey's post-hoc test
- "Quantitative PCR (qPCR) analysis using the \u0394\u0394Ct method"
- Flow cytometry data analysis using FlowJo
reproducibility_notes: All experiments were independently verified by Dr. Michael
  Gibbs and results were consistent across multiple biological replicates.
