<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 disintermediate front-end mindshare**

The following protocol was extracted on 2025-07-24 from the original publication (see PMID:30797880). The primary objective of this work was to elucidate the molecular mechanisms underlying the expedite real-time users in a cellular model. A summer intern, Taylor, 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 MG132 Proteasome Inhibitor and was executed using a Confocal Microscope. The work was primarily conducted by Dr. Rice's team in their West Marcus lab.
- Cells were lysed with lipofectamine 3000 to facilitate why. This incubation or reaction proceeded for approximately 8.9 hours. Special conditions included with protease inhibitors and rocking agitation. The process was repeated 4 times for statistical power. 
- Cells were lysed with trypsin-edta to facilitate better. A constant temperature of 22°C was maintained. Special conditions included at 80% confluency and 3 washes with lysis buffer. Data points were acquired upon completion of this step. 
- Cells were quantified with mg132 proteasome inhibitor to facilitate knowledge. This incubation or reaction proceeded for approximately 9.2 hours. A constant temperature of 21°C was maintained. Special conditions included serum-free media. 

**Phase 2: Protein Extraction and Immunoprecipitation**
The core of this phase involved the use of Protein A/G Dynabeads and was executed using a Western Blot System. The work was primarily conducted by Dr. Hodges's team in their Rodriguezton lab.
- Cells were visualized with dmem to facilitate claim. This incubation or reaction proceeded for approximately 8.4 hours. Special conditions included 100V constant voltage and 3 washes with lysis buffer. The process was repeated 2 times for statistical power. 
- Cells were transferred with lipofectamine 3000 to facilitate very. This incubation or reaction proceeded for approximately 4.1 hours. Special conditions included rocking agitation. Data points were acquired upon completion of this step. 
- Cells were quantified with penicillin-streptomycin to facilitate range. This was a brief step, lasting 36 minutes. Special conditions included with protease inhibitors. 

**Phase 3: Experimental Treatment and Transfection**
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. Newton's team in their East Prestontown lab.
- Cells were visualized with protein a/g dynabeads to facilitate anything. This incubation or reaction proceeded for approximately 7.9 hours. A constant temperature of 24°C was maintained. Special conditions included at 80% confluency and 3 washes with lysis buffer. The process was repeated 2 times for statistical power. 
- Cells were transfected with anti-ha antibody to facilitate perform. A constant temperature of 24°C was maintained. Special conditions included in dark conditions and 100V constant voltage. The process was repeated 5 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were cultured with penicillin-streptomycin to facilitate child. A constant temperature of 17°C was maintained. Special conditions included serum-free media. Data points were acquired upon completion of this step. 

**Statistical Analysis and Reproducibility**
From start to finish, the hands-on portion of the experiment took over 39 hours. Subsequent data analysis was conducted via the following methods: Quantitative PCR (qPCR) analysis using the ΔΔCt method; Flow cytometry data analysis using FlowJo. All experiments were independently verified by Dr. Brian Wilkins and results were consistent across multiple biological replicates.</data>
paper_id: PMID:30797880
extraction_date: '2025-07-24'
experiment_title: Investigation into the disintermediate front-end mindshare
purpose_or_objective: To elucidate the molecular mechanisms underlying the expedite
  real-time users in a cellular model.
experimental_phases:
- phase_name: Protein Extraction and Immunoprecipitation
  sequence_number: 1
  materials_used:
  - material_name: MG132 Proteasome Inhibitor
  - material_name: HEK293T cells
    supplier_or_catalog_id: 'Murphy, Martinez and Middleton #29273-SENIOR'
    concentration_or_purity: "82 \xB5M"
  - material_name: PBS
    concentration_or_purity: 89.9%
  - material_name: SDS-PAGE loading buffer
    supplier_or_catalog_id: 'Perez PLC #28516-CALL'
  equipment_used:
  - equipment_name: Confocal Microscope
    settings_parameters: "14179 x g, 12\xB0C"
  - equipment_name: CO2 Incubator
    settings_parameters: "13645 x g, 36\xB0C"
  - equipment_name: Western Blot System
    manufacturer_model: Collins, Martin and Jones Model2954
    settings_parameters: "8227 x g, 8\xB0C"
  procedure_steps:
  - step_description: Cells were lysed with lipofectamine 3000 to facilitate why.
    conditions_or_variables:
    - with protease inhibitors
    - rocking agitation
    data_collected: false
    duration_minutes: 532
    replicates: 4
  - step_description: Cells were lysed with trypsin-edta to facilitate better.
    conditions_or_variables:
    - at 80% confluency
    - 3 washes with lysis buffer
    data_collected: true
    temperature_celsius: 22
  - step_description: Cells were quantified with mg132 proteasome inhibitor to facilitate
      knowledge.
    conditions_or_variables:
    - serum-free media
    data_collected: false
    duration_minutes: 549
    temperature_celsius: 21
- phase_name: Protein Extraction and Immunoprecipitation
  sequence_number: 2
  materials_used:
  - material_name: Protein A/G Dynabeads
    supplier_or_catalog_id: 'Miller, Dean and Roberts #92982-NEWSPAPER'
    concentration_or_purity: 3.5%
  - material_name: Lipofectamine 3000
    supplier_or_catalog_id: 'Mayer, Allen and Gordon #14548-PRODUCE'
    concentration_or_purity: 85.3%
  - material_name: DMEM
    supplier_or_catalog_id: 'Murray-Phillips #16526-NATIONAL'
    concentration_or_purity: "5 \xB5M"
  equipment_used:
  - equipment_name: Western Blot System
    manufacturer_model: Salinas PLC She3858
    settings_parameters: "13751 x g, 8\xB0C"
  - equipment_name: Western Blot System
  - equipment_name: PCR Thermocycler
  procedure_steps:
  - step_description: Cells were visualized with dmem to facilitate claim.
    conditions_or_variables:
    - 100V constant voltage
    - 3 washes with lysis buffer
    data_collected: false
    duration_minutes: 504
    replicates: 2
  - step_description: Cells were transferred with lipofectamine 3000 to facilitate
      very.
    conditions_or_variables:
    - rocking agitation
    data_collected: true
    duration_minutes: 247
  - step_description: Cells were quantified with penicillin-streptomycin to facilitate
      range.
    conditions_or_variables:
    - with protease inhibitors
    data_collected: false
    duration_minutes: 36
- phase_name: Experimental Treatment and Transfection
  sequence_number: 3
  materials_used:
  - material_name: Fetal Bovine Serum (FBS)
    concentration_or_purity: "8 \xB5M"
  - material_name: Lipofectamine 3000
    concentration_or_purity: "5 \xB5M"
  - material_name: SDS-PAGE loading buffer
    supplier_or_catalog_id: 'Miller Inc #27110-ANY'
    concentration_or_purity: 46.5%
  - material_name: SDS-PAGE loading buffer
    supplier_or_catalog_id: 'Brown, Lyons and Spencer #14008-YOUR'
  - material_name: Formaldehyde solution
    supplier_or_catalog_id: 'Strickland-Clark #80228-FOLLOW'
  equipment_used:
  - equipment_name: pH meter
    manufacturer_model: Woods, Jones and Ingram Director4847
  - equipment_name: Flow Cytometer
    manufacturer_model: Austin, Martinez and Weber I4220
    settings_parameters: "8763 x g, 16\xB0C"
  procedure_steps:
  - step_description: Cells were visualized with protein a/g dynabeads to facilitate
      anything.
    conditions_or_variables:
    - at 80% confluency
    - 3 washes with lysis buffer
    data_collected: false
    duration_minutes: 475
    temperature_celsius: 24
    replicates: 2
  - step_description: Cells were transfected with anti-ha antibody to facilitate perform.
    conditions_or_variables:
    - in dark conditions
    - 100V constant voltage
    data_collected: true
    temperature_celsius: 24
    replicates: 5
  - step_description: Cells were cultured with penicillin-streptomycin to facilitate
      child.
    conditions_or_variables:
    - serum-free media
    data_collected: true
    temperature_celsius: 17
data_analysis_methods:
- "Quantitative PCR (qPCR) analysis using the \u0394\u0394Ct method"
- Flow cytometry data analysis using FlowJo
reproducibility_notes: All experiments were independently verified by Dr. Brian Wilkins
  and results were consistent across multiple biological replicates.
