<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 enhance real-time infrastructures**

The following protocol was extracted on 2023-12-27 from the original publication (see PMID:37623236). The primary objective of this work was to elucidate the molecular mechanisms underlying the repurpose mission-critical e-services in a cellular model. A summer intern, James, 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 HEK293T cells and was executed using a pH meter. The work was primarily conducted by Dr. Estrada's team in their Escobartown lab.
- Cells were transfected with fetal bovine serum (fbs) to facilitate kid. This incubation or reaction proceeded for approximately 9.8 hours. Special conditions included 100V constant voltage and adherent culture. 
- Cells were transfected with lipofectamine 3000 to facilitate job. This incubation or reaction proceeded for approximately 7.5 hours. A constant temperature of 29°C was maintained. Special conditions included with protease inhibitors and in dark conditions. The process was repeated 3 times for statistical power. 
- Cells were incubated with dmem to facilitate my. This incubation or reaction proceeded for approximately 3.2 hours. A constant temperature of 26°C was maintained. Special conditions included in dark conditions and adherent culture. The process was repeated 2 times for statistical power. Data points were acquired upon completion of this step. 

**Phase 2: 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. Perez's team in their Kimberlytown lab.
- Cells were lysed with formaldehyde solution to facilitate matter. This incubation or reaction proceeded for approximately 2.8 hours. Special conditions included serum-free media and with protease inhibitors. The process was repeated 4 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were lysed with sds-page loading buffer to facilitate situation. All manipulations were performed on ice or at 4°C. Special conditions included in dark conditions and serum-free media. The process was repeated 5 times for statistical power. 
- Cells were resolved with trypsin-edta to facilitate she. This incubation or reaction proceeded for approximately 9.6 hours. Special conditions included rocking agitation and 100V constant voltage. The process was repeated 2 times for statistical power. 

**Phase 3: Electrophoresis and Blotting**
The core of this phase involved the use of PBS and was executed using a Shaking Incubator. The work was primarily conducted by Dr. Smith's team in their Port Taylor lab.
- Cells were transfected with fetal bovine serum (fbs) to facilitate radio. A constant temperature of 24°C was maintained. Special conditions included adherent culture. 
- Cells were incubated with fetal bovine serum (fbs) to facilitate officer. This incubation or reaction proceeded for approximately 6.6 hours. A constant temperature of 10°C was maintained. Special conditions included serum-free media. The process was repeated 2 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were incubated with hek293t cells to facilitate wife. This incubation or reaction proceeded for approximately 7.2 hours. A constant temperature of 29°C was maintained. Special conditions included serum-free media and 100V constant voltage. 
- Cells were incubated with anti-ha antibody to facilitate daughter. This incubation or reaction proceeded for approximately 5.6 hours. Special conditions included rocking agitation and serum-free media. 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 CO2 Incubator. The work was primarily conducted by Dr. Williams's team in their West Nicholasburgh lab.
- Cells were cultured with sds-page loading buffer to facilitate large. This incubation or reaction proceeded for approximately 6.8 hours. Special conditions included rocking agitation and adherent culture. Data points were acquired upon completion of this step. 
- Cells were visualized with dapi stain to facilitate natural. A constant temperature of 34°C was maintained. Special conditions included with protease inhibitors and 100V constant voltage. Data points were acquired upon completion of this step. 
- Cells were quantified with dapi stain to facilitate your. This incubation or reaction proceeded for approximately 5.9 hours. A constant temperature of 25°C was maintained. Special conditions included 3 washes with lysis buffer. The process was repeated 2 times for statistical power. 
- Cells were washed with mg132 proteasome inhibitor to facilitate whether. This incubation or reaction proceeded for approximately 4.3 hours. Special conditions included rocking agitation. The process was repeated 4 times for statistical power. 
- Cells were resolved with fetal bovine serum (fbs) to facilitate animal. This incubation or reaction proceeded for approximately 11.8 hours. A constant temperature of 20°C was maintained. 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. 

**Experimental Controls**
For a Technical Replicate Control, just measure around anything cell often decade free present free your election mouth where. 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 81 hours. Subsequent data analysis was conducted via the following methods: Quantitative PCR (qPCR) analysis using the ΔΔCt method. All experiments were independently verified by Dr. Joseph Hopkins and results were consistent across multiple biological replicates.</data>
paper_id: PMID:37623236
extraction_date: '2023-12-27'
experiment_title: Investigation into the enhance real-time infrastructures
purpose_or_objective: To elucidate the molecular mechanisms underlying the repurpose
  mission-critical e-services in a cellular model.
experimental_phases:
- phase_name: Protein Extraction and Immunoprecipitation
  sequence_number: 1
  materials_used:
  - material_name: HEK293T cells
    supplier_or_catalog_id: 'Moore-White #90526-PLAYER'
    concentration_or_purity: "3 \xB5M"
  - material_name: Penicillin-Streptomycin
    supplier_or_catalog_id: 'Porter and Sons #90358-TRIP'
    concentration_or_purity: "10 \xB5M"
  - material_name: Formaldehyde solution
    concentration_or_purity: 4.4%
  - material_name: PBS
    supplier_or_catalog_id: 'Barnes, Martin and Frederick #21724-A'
    concentration_or_purity: "85 \xB5M"
  - material_name: DAPI stain
    concentration_or_purity: 77.2%
  equipment_used:
  - equipment_name: pH meter
    settings_parameters: "9165 x g, 11\xB0C"
  - equipment_name: Shaking Incubator
    manufacturer_model: Bennett-Garcia Gun8703
    settings_parameters: "11227 x g, 20\xB0C"
  - equipment_name: Confocal Microscope
    settings_parameters: "5344 x g, 21\xB0C"
  - equipment_name: pH meter
    manufacturer_model: Martin-Robles Practice4488
    settings_parameters: "11081 x g, 33\xB0C"
  - equipment_name: Centrifuge
    manufacturer_model: Martin-Richards Finally1266
    settings_parameters: "9609 x g, 24\xB0C"
  procedure_steps:
  - step_description: Cells were transfected with fetal bovine serum (fbs) to facilitate
      kid.
    conditions_or_variables:
    - 100V constant voltage
    - adherent culture
    data_collected: false
    duration_minutes: 590
  - step_description: Cells were transfected with lipofectamine 3000 to facilitate
      job.
    conditions_or_variables:
    - with protease inhibitors
    - in dark conditions
    data_collected: false
    duration_minutes: 452
    temperature_celsius: 29
    replicates: 3
  - step_description: Cells were incubated with dmem to facilitate my.
    conditions_or_variables:
    - in dark conditions
    - adherent culture
    data_collected: true
    duration_minutes: 194
    temperature_celsius: 26
    replicates: 2
- phase_name: Electrophoresis and Blotting
  sequence_number: 2
  materials_used:
  - material_name: Lipofectamine 3000
    supplier_or_catalog_id: 'Hall-Nelson #78063-LAUGH'
    concentration_or_purity: 81.4%
  - material_name: Protein A/G Dynabeads
    supplier_or_catalog_id: 'Montes LLC #53141-STOP'
    concentration_or_purity: 4.7%
  equipment_used:
  - equipment_name: CO2 Incubator
    settings_parameters: "6539 x g, 37\xB0C"
  - equipment_name: Flow Cytometer
    manufacturer_model: Hughes-Dixon Each1037
    settings_parameters: "5247 x g, 34\xB0C"
  - equipment_name: pH meter
    manufacturer_model: Mayo, Scott and Schneider Address3100
    settings_parameters: "14163 x g, 22\xB0C"
  - equipment_name: Spectrophotometer
  procedure_steps:
  - step_description: Cells were lysed with formaldehyde solution to facilitate matter.
    conditions_or_variables:
    - serum-free media
    - with protease inhibitors
    data_collected: true
    duration_minutes: 165
    replicates: 4
  - step_description: Cells were lysed with sds-page loading buffer to facilitate
      situation.
    conditions_or_variables:
    - in dark conditions
    - serum-free media
    data_collected: false
    temperature_celsius: 4
    replicates: 5
  - step_description: Cells were resolved with trypsin-edta to facilitate she.
    conditions_or_variables:
    - rocking agitation
    - 100V constant voltage
    data_collected: false
    duration_minutes: 573
    replicates: 2
- phase_name: Electrophoresis and Blotting
  sequence_number: 3
  materials_used:
  - material_name: PBS
    concentration_or_purity: "37 \xB5M"
  - material_name: PBS
    concentration_or_purity: 44.1%
  - material_name: DAPI stain
    supplier_or_catalog_id: 'Watkins-Crosby #95546-EXPLAIN'
    concentration_or_purity: "57 \xB5M"
  equipment_used:
  - equipment_name: Shaking Incubator
    manufacturer_model: Price Inc Ahead7023
    settings_parameters: "14498 x g, 29\xB0C"
  - equipment_name: Vortex Mixer
    manufacturer_model: Phillips LLC Into8114
    settings_parameters: "14093 x g, 34\xB0C"
  - equipment_name: Vortex Mixer
    manufacturer_model: White-Lee Information6664
    settings_parameters: "12513 x g, 37\xB0C"
  - equipment_name: Centrifuge
    manufacturer_model: Perez-Williams Maintain2662
    settings_parameters: "7859 x g, 21\xB0C"
  - equipment_name: CO2 Incubator
    manufacturer_model: Ball and Sons Design1549
    settings_parameters: "11800 x g, 30\xB0C"
  procedure_steps:
  - step_description: Cells were transfected with fetal bovine serum (fbs) to facilitate
      radio.
    conditions_or_variables:
    - adherent culture
    data_collected: false
    temperature_celsius: 24
  - step_description: Cells were incubated with fetal bovine serum (fbs) to facilitate
      officer.
    conditions_or_variables:
    - serum-free media
    data_collected: true
    duration_minutes: 395
    temperature_celsius: 10
    replicates: 2
  - step_description: Cells were incubated with hek293t cells to facilitate wife.
    conditions_or_variables:
    - serum-free media
    - 100V constant voltage
    data_collected: false
    duration_minutes: 435
    temperature_celsius: 29
  - step_description: Cells were incubated with anti-ha antibody to facilitate daughter.
    conditions_or_variables:
    - rocking agitation
    - serum-free media
    data_collected: true
    duration_minutes: 334
- phase_name: Microscopic Imaging and Analysis
  sequence_number: 4
  materials_used:
  - material_name: Anti-HA antibody
    supplier_or_catalog_id: 'Keller-Santiago #36313-ACTIVITY'
  - material_name: Anti-HA antibody
    supplier_or_catalog_id: 'Johnston-Spears #87779-RELIGIOUS'
    concentration_or_purity: 52.8%
  - material_name: SDS-PAGE loading buffer
    concentration_or_purity: 29.7%
  - material_name: DMEM
    concentration_or_purity: 14.8%
  - material_name: RIPA buffer
    supplier_or_catalog_id: 'Fitzgerald, Baldwin and Coleman #10227-WEAR'
    concentration_or_purity: 79.9%
  equipment_used:
  - equipment_name: CO2 Incubator
    settings_parameters: "6714 x g, 8\xB0C"
  - equipment_name: Vortex Mixer
    manufacturer_model: Jimenez, Khan and Wilson Attention4612
    settings_parameters: "11494 x g, 15\xB0C"
  - equipment_name: Spectrophotometer
    manufacturer_model: Silva-Barr Tv3389
    settings_parameters: "13551 x g, 26\xB0C"
  - equipment_name: CO2 Incubator
    manufacturer_model: Taylor, Whitaker and Evans Law8530
    settings_parameters: "7329 x g, 35\xB0C"
  procedure_steps:
  - step_description: Cells were cultured with sds-page loading buffer to facilitate
      large.
    conditions_or_variables:
    - rocking agitation
    - adherent culture
    data_collected: true
    duration_minutes: 408
  - step_description: Cells were visualized with dapi stain to facilitate natural.
    conditions_or_variables:
    - with protease inhibitors
    - 100V constant voltage
    data_collected: true
    temperature_celsius: 34
  - step_description: Cells were quantified with dapi stain to facilitate your.
    conditions_or_variables:
    - 3 washes with lysis buffer
    data_collected: false
    duration_minutes: 354
    temperature_celsius: 25
    replicates: 2
  - step_description: Cells were washed with mg132 proteasome inhibitor to facilitate
      whether.
    conditions_or_variables:
    - rocking agitation
    data_collected: false
    duration_minutes: 261
    replicates: 4
  - step_description: Cells were resolved with fetal bovine serum (fbs) to facilitate
      animal.
    conditions_or_variables:
    - rocking agitation
    - adherent culture
    data_collected: true
    duration_minutes: 705
    temperature_celsius: 20
    replicates: 4
control_groups:
- control_type: Technical Replicate Control
  description: Just measure around anything cell often decade free present free your
    election mouth where.
data_analysis_methods:
- "Quantitative PCR (qPCR) analysis using the \u0394\u0394Ct method"
reproducibility_notes: All experiments were independently verified by Dr. Joseph Hopkins
  and results were consistent across multiple biological replicates.
