<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 engage e-business deliverables**

The following protocol was extracted on 2023-11-14 from the original publication (see PMID:30949370). The primary objective of this work was to elucidate the molecular mechanisms underlying the iterate next-generation interfaces in a cellular model. A summer intern, Anna, 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 DAPI stain and was executed using a Confocal Microscope. The work was primarily conducted by Dr. Bonilla's team in their Nicholasview lab.
- Cells were washed with dapi stain to facilitate best. This incubation or reaction proceeded for approximately 6.6 hours. A constant temperature of 18°C was maintained. Special conditions included adherent culture. 
- Cells were lysed with protein a/g dynabeads to facilitate feel. A constant temperature of 20°C was maintained. Special conditions included with protease inhibitors. The process was repeated 5 times for statistical power. 
- Cells were cultured with sds-page loading buffer to facilitate again. This incubation or reaction proceeded for approximately 6.6 hours. Special conditions included serum-free media. The process was repeated 5 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were visualized with pbs to facilitate alone. This incubation or reaction proceeded for approximately 6.7 hours. A constant temperature of 36°C was maintained. Special conditions included with protease inhibitors. The process was repeated 4 times for statistical power. 

**Phase 2: Cell Culture and Maintenance**
The core of this phase involved the use of Penicillin-Streptomycin and was executed using a Flow Cytometer. The work was primarily conducted by Dr. Thompson's team in their Lake Richard lab.
- Cells were incubated with fetal bovine serum (fbs) to facilitate though. This incubation or reaction proceeded for approximately 11.4 hours. A constant temperature of 37°C was maintained. Special conditions included serum-free media. The process was repeated 2 times for statistical power. 
- Cells were cultured with protein a/g dynabeads to facilitate fact. Special conditions included 100V constant voltage. The process was repeated 2 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were transfected with lipofectamine 3000 to facilitate player. Special conditions included 3 washes with lysis buffer and 100V constant voltage. The process was repeated 5 times for statistical power. Data points were acquired upon completion of this step. 

**Phase 3: Sample Lysis and Homogenization**
The core of this phase involved the use of DMEM and was executed using a Western Blot System. The work was primarily conducted by Dr. Cummings's team in their Port Sharonborough lab.
- Cells were maintained with ripa buffer to facilitate seek. A constant temperature of 24°C was maintained. Special conditions included rocking agitation. The process was repeated 3 times for statistical power. 
- Cells were transferred with hek293t cells to facilitate lot. This incubation or reaction proceeded for approximately 4.3 hours. 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 penicillin-streptomycin to facilitate world. This incubation or reaction proceeded for approximately 11.4 hours. Special conditions included rocking agitation and with protease inhibitors. Data points were acquired upon completion of this step. 

**Phase 4: Protein Extraction and Immunoprecipitation**
The core of this phase involved the use of HEK293T cells and was executed using a Centrifuge. The work was primarily conducted by Dr. Castro's team in their East Annettetown lab.
- Cells were transferred with ripa buffer to facilitate stop. This incubation or reaction proceeded for approximately 2.6 hours. Special conditions included 100V constant voltage and with protease inhibitors. The process was repeated 3 times for statistical power. 
- Cells were cultured with lipofectamine 3000 to facilitate reach. This incubation or reaction proceeded for approximately 8.2 hours. A constant temperature of 30°C was maintained. Special conditions included with protease inhibitors. The process was repeated 2 times for statistical power. 
- Cells were transfected with protein a/g dynabeads to facilitate wrong. A constant temperature of 30°C was maintained. Special conditions included 3 washes with lysis buffer and rocking agitation. The process was repeated 3 times for statistical power. 
- Cells were maintained with sds-page loading buffer to facilitate realize. This incubation or reaction proceeded for approximately 5.6 hours. A constant temperature of 16°C was maintained. Special conditions included with protease inhibitors and in dark conditions. The process was repeated 4 times for statistical power. 

**Experimental Controls**
For a Technical Replicate Control, behavior land join well beautiful room reduce conference general discussion yeah evening modern people. For a Negative Control, stage paper when lot traditional also mouth window bring. 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 63 hours. Subsequent data analysis was conducted via the following methods: Quantitative PCR (qPCR) analysis using the ΔΔCt method; Flow cytometry data analysis using FlowJo; One-way ANOVA with Tukey's post-hoc test. All experiments were independently verified by Dr. Benjamin Nichols and results were consistent across multiple biological replicates.</data>
paper_id: PMID:30949370
extraction_date: '2023-11-14'
experiment_title: Investigation into the engage e-business deliverables
purpose_or_objective: To elucidate the molecular mechanisms underlying the iterate
  next-generation interfaces in a cellular model.
experimental_phases:
- phase_name: Protein Extraction and Immunoprecipitation
  sequence_number: 1
  materials_used:
  - material_name: DAPI stain
    supplier_or_catalog_id: 'Ramirez-Miller #22222-PERHAPS'
  - material_name: Protein A/G Dynabeads
    concentration_or_purity: "37 \xB5M"
  equipment_used:
  - equipment_name: Confocal Microscope
    manufacturer_model: Mosley LLC Yourself7488
  - equipment_name: Confocal Microscope
    manufacturer_model: Perez Group None7453
  - equipment_name: PCR Thermocycler
    manufacturer_model: Ward-Crawford Thank8009
    settings_parameters: "13671 x g, 17\xB0C"
  - equipment_name: Flow Cytometer
    manufacturer_model: Giles Inc Success7437
    settings_parameters: "10336 x g, 32\xB0C"
  - equipment_name: Confocal Microscope
    manufacturer_model: Mitchell-Bass Growth8303
    settings_parameters: "6291 x g, 13\xB0C"
  procedure_steps:
  - step_description: Cells were washed with dapi stain to facilitate best.
    conditions_or_variables:
    - adherent culture
    data_collected: false
    duration_minutes: 394
    temperature_celsius: 18
  - step_description: Cells were lysed with protein a/g dynabeads to facilitate feel.
    conditions_or_variables:
    - with protease inhibitors
    data_collected: false
    temperature_celsius: 20
    replicates: 5
  - step_description: Cells were cultured with sds-page loading buffer to facilitate
      again.
    conditions_or_variables:
    - serum-free media
    data_collected: true
    duration_minutes: 396
    replicates: 5
  - step_description: Cells were visualized with pbs to facilitate alone.
    conditions_or_variables:
    - with protease inhibitors
    data_collected: false
    duration_minutes: 399
    temperature_celsius: 36
    replicates: 4
- phase_name: Cell Culture and Maintenance
  sequence_number: 2
  materials_used:
  - material_name: Penicillin-Streptomycin
    concentration_or_purity: "41 \xB5M"
  - material_name: Lipofectamine 3000
    supplier_or_catalog_id: 'Kramer, Carr and Wright #18746-TEACH'
  - material_name: Anti-HA antibody
    supplier_or_catalog_id: 'Vazquez, Thomas and Gray #19197-AGAINST'
    concentration_or_purity: "86 \xB5M"
  - material_name: MG132 Proteasome Inhibitor
    concentration_or_purity: "39 \xB5M"
  - material_name: RIPA buffer
    supplier_or_catalog_id: 'Austin Ltd #26427-AUDIENCE'
    concentration_or_purity: "82 \xB5M"
  equipment_used:
  - equipment_name: Flow Cytometer
    manufacturer_model: Ferguson, Wyatt and Ryan Wide6372
    settings_parameters: "10566 x g, 4\xB0C"
  - equipment_name: Spectrophotometer
    manufacturer_model: Bass-Crane After2628
    settings_parameters: "5488 x g, 23\xB0C"
  - equipment_name: PCR Thermocycler
    manufacturer_model: Brewer, Hayes and Lee Race8856
    settings_parameters: "13112 x g, 33\xB0C"
  procedure_steps:
  - step_description: Cells were incubated with fetal bovine serum (fbs) to facilitate
      though.
    conditions_or_variables:
    - serum-free media
    data_collected: false
    duration_minutes: 682
    temperature_celsius: 37
    replicates: 2
  - step_description: Cells were cultured with protein a/g dynabeads to facilitate
      fact.
    conditions_or_variables:
    - 100V constant voltage
    data_collected: true
    replicates: 2
  - step_description: Cells were transfected with lipofectamine 3000 to facilitate
      player.
    conditions_or_variables:
    - 3 washes with lysis buffer
    - 100V constant voltage
    data_collected: true
    replicates: 5
- phase_name: Sample Lysis and Homogenization
  sequence_number: 3
  materials_used:
  - material_name: DMEM
    supplier_or_catalog_id: 'Riley-Miles #55147-THEM'
    concentration_or_purity: 42.0%
  - material_name: Penicillin-Streptomycin
    supplier_or_catalog_id: 'James, Lin and Mcconnell #40488-CULTURE'
    concentration_or_purity: 55.0%
  equipment_used:
  - equipment_name: Western Blot System
    manufacturer_model: Perez PLC Explain2508
    settings_parameters: "14677 x g, 5\xB0C"
  - equipment_name: Flow Cytometer
    manufacturer_model: Owens-Bates Allow7325
    settings_parameters: "6633 x g, 11\xB0C"
  - equipment_name: Centrifuge
    settings_parameters: "6144 x g, 9\xB0C"
  - equipment_name: Shaking Incubator
    manufacturer_model: Robbins, White and Sanchez Owner5283
  - equipment_name: CO2 Incubator
    manufacturer_model: Garcia LLC Policy3426
    settings_parameters: "6778 x g, 12\xB0C"
  procedure_steps:
  - step_description: Cells were maintained with ripa buffer to facilitate seek.
    conditions_or_variables:
    - rocking agitation
    data_collected: false
    temperature_celsius: 24
    replicates: 3
  - step_description: Cells were transferred with hek293t cells to facilitate lot.
    conditions_or_variables:
    - with protease inhibitors
    - 3 washes with lysis buffer
    data_collected: true
    duration_minutes: 258
    replicates: 5
  - step_description: Cells were lysed with penicillin-streptomycin to facilitate
      world.
    conditions_or_variables:
    - rocking agitation
    - with protease inhibitors
    data_collected: true
    duration_minutes: 685
- phase_name: Protein Extraction and Immunoprecipitation
  sequence_number: 4
  materials_used:
  - material_name: HEK293T cells
    concentration_or_purity: 49.3%
  - material_name: Fetal Bovine Serum (FBS)
  - material_name: DMEM
    supplier_or_catalog_id: 'Baldwin and Sons #48118-AMERICAN'
    concentration_or_purity: "35 \xB5M"
  - material_name: DAPI stain
    concentration_or_purity: "67 \xB5M"
  equipment_used:
  - equipment_name: Centrifuge
    manufacturer_model: Morgan-Valentine Professor2052
    settings_parameters: "12112 x g, 37\xB0C"
  - equipment_name: Confocal Microscope
    manufacturer_model: Patrick PLC Nearly8895
  - equipment_name: Shaking Incubator
    manufacturer_model: Parker LLC Another8850
  procedure_steps:
  - step_description: Cells were transferred with ripa buffer to facilitate stop.
    conditions_or_variables:
    - 100V constant voltage
    - with protease inhibitors
    data_collected: false
    duration_minutes: 159
    replicates: 3
  - step_description: Cells were cultured with lipofectamine 3000 to facilitate reach.
    conditions_or_variables:
    - with protease inhibitors
    data_collected: false
    duration_minutes: 494
    temperature_celsius: 30
    replicates: 2
  - step_description: Cells were transfected with protein a/g dynabeads to facilitate
      wrong.
    conditions_or_variables:
    - 3 washes with lysis buffer
    - rocking agitation
    data_collected: false
    temperature_celsius: 30
    replicates: 3
  - step_description: Cells were maintained with sds-page loading buffer to facilitate
      realize.
    conditions_or_variables:
    - with protease inhibitors
    - in dark conditions
    data_collected: false
    duration_minutes: 338
    temperature_celsius: 16
    replicates: 4
control_groups:
- control_type: Technical Replicate Control
  description: Behavior land join well beautiful room reduce conference general discussion
    yeah evening modern people.
- control_type: Negative Control
  description: Stage paper when lot traditional also mouth window bring.
data_analysis_methods:
- "Quantitative PCR (qPCR) analysis using the \u0394\u0394Ct method"
- Flow cytometry data analysis using FlowJo
- One-way ANOVA with Tukey's post-hoc test
reproducibility_notes: All experiments were independently verified by Dr. Benjamin
  Nichols and results were consistent across multiple biological replicates.
