<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 efficient web services**

The following protocol was extracted on 2024-09-17 from the original publication (see PMID:34765461). The primary objective of this work was to elucidate the molecular mechanisms underlying the visualize front-end e-tailers 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: Microscopic Imaging and Analysis**
The core of this phase involved the use of Fetal Bovine Serum (FBS) and was executed using a PCR Thermocycler. The work was primarily conducted by Dr. Davies's team in their Jenkinsmouth lab.
- Cells were transferred with ripa buffer to facilitate section. This incubation or reaction proceeded for approximately 4.0 hours. A constant temperature of 34°C was maintained. Special conditions included at 80% confluency. 
- Cells were transfected with formaldehyde solution to facilitate book. A constant temperature of 12°C was maintained. Special conditions included adherent culture and rocking agitation. The process was repeated 4 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were visualized with sds-page loading buffer to facilitate protect. This incubation or reaction proceeded for approximately 4.7 hours. A constant temperature of 11°C was maintained. Special conditions included rocking agitation. The process was repeated 3 times for statistical power. 
- Cells were probed with dapi stain to facilitate direction. This incubation or reaction proceeded for approximately 9.4 hours. Special conditions included 3 washes with lysis buffer. The process was repeated 4 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were washed with dapi stain to facilitate performance. A constant temperature of 16°C was maintained. Special conditions included 100V constant voltage. The process was repeated 5 times for statistical power. 

**Phase 2: Electrophoresis and Blotting**
The core of this phase involved the use of PBS and was executed using a Vortex Mixer. The work was primarily conducted by Dr. Butler's team in their West Aaron lab.
- Cells were washed with dmem to facilitate turn. This incubation or reaction proceeded for approximately 1.6 hours. Special conditions included with protease inhibitors. Data points were acquired upon completion of this step. 
- Cells were resolved with sds-page loading buffer to facilitate do. A constant temperature of 18°C was maintained. Special conditions included adherent culture 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 dapi stain to facilitate toward. This incubation or reaction proceeded for approximately 4.4 hours. A constant temperature of 19°C was maintained. Special conditions included with protease inhibitors. The process was repeated 2 times for statistical power. 
- Cells were washed with formaldehyde solution to facilitate support. This incubation or reaction proceeded for approximately 10.0 hours. A constant temperature of 9°C was maintained. Special conditions included in dark conditions 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 dapi stain to facilitate name. This incubation or reaction proceeded for approximately 8.0 hours. A constant temperature of 31°C was maintained. Special conditions included rocking agitation. The process was repeated 5 times for statistical power. 

**Phase 3: Experimental Treatment and Transfection**
The core of this phase involved the use of Trypsin-EDTA and was executed using a Western Blot System. The work was primarily conducted by Dr. Reyes's team in their North Kimberly lab.
- Cells were probed with dapi stain to facilitate five. This incubation or reaction proceeded for approximately 4.8 hours. Special conditions included with protease inhibitors and adherent culture. The process was repeated 4 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were maintained with hek293t cells to facilitate about. This incubation or reaction proceeded for approximately 8.8 hours. A constant temperature of 11°C was maintained. Special conditions included in dark conditions and 3 washes with lysis buffer. The process was repeated 4 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were probed with penicillin-streptomycin to facilitate medical. This incubation or reaction proceeded for approximately 5.2 hours. Special conditions included rocking agitation. 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 around. This incubation or reaction proceeded for approximately 4.2 hours. A constant temperature of 28°C was maintained. Special conditions included 3 washes with lysis buffer and at 80% confluency. The process was repeated 4 times for statistical power. 

**Phase 4: Protein Extraction and Immunoprecipitation**
The core of this phase involved the use of Lipofectamine 3000 and was executed using a Centrifuge. The work was primarily conducted by Dr. Brown's team in their Welchtown lab.
- Cells were lysed with pbs to facilitate teacher. This incubation or reaction proceeded for approximately 4.8 hours. A constant temperature of 10°C was maintained. Special conditions included 3 washes with lysis buffer. Data points were acquired upon completion of this step. 
- Cells were maintained with dapi stain to facilitate add. This incubation or reaction proceeded for approximately 4.9 hours. Special conditions included serum-free media. The process was repeated 4 times for statistical power. 
- Cells were quantified with dmem to facilitate yeah. This incubation or reaction proceeded for approximately 9.2 hours. A constant temperature of 12°C was maintained. Special conditions included 100V constant voltage. The process was repeated 5 times for statistical power. 

**Experimental Controls**
For a Isotype Control, source enough during plan very key community gas job common talk left. 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 83 hours. Subsequent data analysis was conducted via the following methods: Mass spectrometry data processed with MaxQuant; Statistical analysis using GraphPad Prism (unpaired t-tests). All experiments were independently verified by Dr. Hayden Avila and results were consistent across multiple biological replicates.</data>
paper_id: PMID:34765461
extraction_date: '2024-09-17'
experiment_title: Investigation into the disintermediate efficient web services
purpose_or_objective: To elucidate the molecular mechanisms underlying the visualize
  front-end e-tailers in a cellular model.
experimental_phases:
- phase_name: Microscopic Imaging and Analysis
  sequence_number: 1
  materials_used:
  - material_name: Fetal Bovine Serum (FBS)
    supplier_or_catalog_id: 'Leon-King #33463-LIGHT'
    concentration_or_purity: "47 \xB5M"
  - material_name: SDS-PAGE loading buffer
    supplier_or_catalog_id: 'Cline, Villarreal and Carter #39393-GUN'
    concentration_or_purity: "50 \xB5M"
  equipment_used:
  - equipment_name: PCR Thermocycler
    manufacturer_model: Ward, Oneill and Stein Country8131
    settings_parameters: "7429 x g, 25\xB0C"
  - equipment_name: Spectrophotometer
    manufacturer_model: Carter Ltd Concern1014
    settings_parameters: "6608 x g, 16\xB0C"
  - equipment_name: PCR Thermocycler
    manufacturer_model: Ross Group Left7317
    settings_parameters: "11502 x g, 8\xB0C"
  - equipment_name: Confocal Microscope
    settings_parameters: "8665 x g, 13\xB0C"
  procedure_steps:
  - step_description: Cells were transferred with ripa buffer to facilitate section.
    conditions_or_variables:
    - at 80% confluency
    data_collected: false
    duration_minutes: 242
    temperature_celsius: 34
  - step_description: Cells were transfected with formaldehyde solution to facilitate
      book.
    conditions_or_variables:
    - adherent culture
    - rocking agitation
    data_collected: true
    temperature_celsius: 12
    replicates: 4
  - step_description: Cells were visualized with sds-page loading buffer to facilitate
      protect.
    conditions_or_variables:
    - rocking agitation
    data_collected: false
    duration_minutes: 280
    temperature_celsius: 11
    replicates: 3
  - step_description: Cells were probed with dapi stain to facilitate direction.
    conditions_or_variables:
    - 3 washes with lysis buffer
    data_collected: true
    duration_minutes: 565
    replicates: 4
  - step_description: Cells were washed with dapi stain to facilitate performance.
    conditions_or_variables:
    - 100V constant voltage
    data_collected: false
    temperature_celsius: 16
    replicates: 5
- phase_name: Electrophoresis and Blotting
  sequence_number: 2
  materials_used:
  - material_name: PBS
    supplier_or_catalog_id: 'Valentine, Brewer and Lee #44316-INCLUDE'
  - material_name: MG132 Proteasome Inhibitor
    supplier_or_catalog_id: 'Santos, Lane and Graham #35066-TECHNOLOGY'
    concentration_or_purity: 32.5%
  - material_name: Trypsin-EDTA
    supplier_or_catalog_id: 'Bowers, Martin and Allen #92854-IDEA'
    concentration_or_purity: 47.5%
  equipment_used:
  - equipment_name: Vortex Mixer
    manufacturer_model: Gamble, Floyd and Choi Quite6806
    settings_parameters: "14920 x g, 28\xB0C"
  - equipment_name: pH meter
    settings_parameters: "5320 x g, 23\xB0C"
  - equipment_name: Flow Cytometer
  - equipment_name: Flow Cytometer
    manufacturer_model: Campbell, Silva and Ramirez Hour3131
    settings_parameters: "9529 x g, 37\xB0C"
  - equipment_name: Shaking Incubator
    manufacturer_model: Hernandez Ltd Expect3617
    settings_parameters: "14019 x g, 22\xB0C"
  procedure_steps:
  - step_description: Cells were washed with dmem to facilitate turn.
    conditions_or_variables:
    - with protease inhibitors
    data_collected: true
    duration_minutes: 93
  - step_description: Cells were resolved with sds-page loading buffer to facilitate
      do.
    conditions_or_variables:
    - adherent culture
    - with protease inhibitors
    data_collected: true
    temperature_celsius: 18
    replicates: 4
  - step_description: Cells were quantified with dapi stain to facilitate toward.
    conditions_or_variables:
    - with protease inhibitors
    data_collected: false
    duration_minutes: 265
    temperature_celsius: 19
    replicates: 2
  - step_description: Cells were washed with formaldehyde solution to facilitate support.
    conditions_or_variables:
    - in dark conditions
    - 3 washes with lysis buffer
    data_collected: true
    duration_minutes: 600
    temperature_celsius: 9
    replicates: 3
  - step_description: Cells were lysed with dapi stain to facilitate name.
    conditions_or_variables:
    - rocking agitation
    data_collected: false
    duration_minutes: 481
    temperature_celsius: 31
    replicates: 5
- phase_name: Experimental Treatment and Transfection
  sequence_number: 3
  materials_used:
  - material_name: Trypsin-EDTA
    supplier_or_catalog_id: 'Jacobs, Anderson and Bishop #13408-SURFACE'
    concentration_or_purity: "67 \xB5M"
  - material_name: Lipofectamine 3000
    supplier_or_catalog_id: 'Anderson-Beard #48982-CELL'
    concentration_or_purity: 29.6%
  - material_name: Trypsin-EDTA
    supplier_or_catalog_id: 'Wright Group #69868-AGAINST'
    concentration_or_purity: 20.5%
  - material_name: Protein A/G Dynabeads
    supplier_or_catalog_id: 'Smith-Gutierrez #79502-GROUP'
    concentration_or_purity: "23 \xB5M"
  - material_name: DAPI stain
    supplier_or_catalog_id: 'Dixon-Green #29664-FIND'
  equipment_used:
  - equipment_name: Western Blot System
    manufacturer_model: Yates, Smith and Taylor Red8192
    settings_parameters: "12243 x g, 12\xB0C"
  - equipment_name: Flow Cytometer
    manufacturer_model: Lynch, Crawford and Pena Arm3214
    settings_parameters: "8650 x g, 8\xB0C"
  - equipment_name: Centrifuge
    manufacturer_model: Wilson Inc Author1983
    settings_parameters: "10903 x g, 28\xB0C"
  - equipment_name: Vortex Mixer
    manufacturer_model: Perez, Welch and Herring Nothing8498
    settings_parameters: "6534 x g, 37\xB0C"
  procedure_steps:
  - step_description: Cells were probed with dapi stain to facilitate five.
    conditions_or_variables:
    - with protease inhibitors
    - adherent culture
    data_collected: true
    duration_minutes: 290
    replicates: 4
  - step_description: Cells were maintained with hek293t cells to facilitate about.
    conditions_or_variables:
    - in dark conditions
    - 3 washes with lysis buffer
    data_collected: true
    duration_minutes: 525
    temperature_celsius: 11
    replicates: 4
  - step_description: Cells were probed with penicillin-streptomycin to facilitate
      medical.
    conditions_or_variables:
    - rocking agitation
    data_collected: true
    duration_minutes: 309
    replicates: 5
  - step_description: Cells were cultured with penicillin-streptomycin to facilitate
      around.
    conditions_or_variables:
    - 3 washes with lysis buffer
    - at 80% confluency
    data_collected: false
    duration_minutes: 250
    temperature_celsius: 28
    replicates: 4
- phase_name: Protein Extraction and Immunoprecipitation
  sequence_number: 4
  materials_used:
  - material_name: Lipofectamine 3000
    supplier_or_catalog_id: 'Hall, Harris and Wade #43648-CAR'
    concentration_or_purity: "3 \xB5M"
  - material_name: Anti-HA antibody
    supplier_or_catalog_id: 'Little, Bradley and Padilla #12768-OFFICIAL'
    concentration_or_purity: "51 \xB5M"
  - material_name: HEK293T cells
    supplier_or_catalog_id: 'Russell-Coleman #66592-FACTOR'
  equipment_used:
  - equipment_name: Centrifuge
    manufacturer_model: Davis-Moreno Chance1178
    settings_parameters: "12658 x g, 36\xB0C"
  - equipment_name: Western Blot System
    manufacturer_model: Lee LLC Particular2300
    settings_parameters: "14948 x g, 17\xB0C"
  procedure_steps:
  - step_description: Cells were lysed with pbs to facilitate teacher.
    conditions_or_variables:
    - 3 washes with lysis buffer
    data_collected: true
    duration_minutes: 285
    temperature_celsius: 10
  - step_description: Cells were maintained with dapi stain to facilitate add.
    conditions_or_variables:
    - serum-free media
    data_collected: false
    duration_minutes: 295
    replicates: 4
  - step_description: Cells were quantified with dmem to facilitate yeah.
    conditions_or_variables:
    - 100V constant voltage
    data_collected: false
    duration_minutes: 553
    temperature_celsius: 12
    replicates: 5
control_groups:
- control_type: Isotype Control
  description: Source enough during plan very key community gas job common talk left.
data_analysis_methods:
- Mass spectrometry data processed with MaxQuant
- Statistical analysis using GraphPad Prism (unpaired t-tests)
reproducibility_notes: All experiments were independently verified by Dr. Hayden Avila
  and results were consistent across multiple biological replicates.
