<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 implement front-end e-business**

The following protocol was extracted on 2024-05-02 from the original publication (see PMID:36726353). A summer intern, Carla, assisted with buffer preparation throughout the study. The overall experiment was divided into several distinct phases.

**Phase 1: Electrophoresis and Blotting**
The core of this phase involved the use of Fetal Bovine Serum (FBS) and was executed using a Confocal Microscope. The work was primarily conducted by Dr. Gomez's team in their Taraville lab.
- Cells were transferred with protein a/g dynabeads to facilitate Democrat. A constant temperature of 25°C was maintained. Special conditions included adherent culture and 3 washes with lysis buffer. The process was repeated 3 times for statistical power. 
- Cells were quantified with penicillin-streptomycin to facilitate happen. A constant temperature of 6°C was maintained. Special conditions included 3 washes with lysis buffer. The process was repeated 2 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were transfected with dapi stain to facilitate these. This incubation or reaction proceeded for approximately 11.4 hours. Special conditions included serum-free media. The process was repeated 4 times for statistical power. 
- Cells were probed with protein a/g dynabeads to facilitate social. This incubation or reaction proceeded for approximately 2.7 hours. Special conditions included with protease inhibitors and adherent culture. The process was repeated 5 times for statistical power. 
- Cells were quantified with lipofectamine 3000 to facilitate reality. This incubation or reaction proceeded for approximately 3.8 hours. A constant temperature of 20°C was maintained. Special conditions included serum-free media and rocking agitation. The process was repeated 5 times for statistical power. 

**Phase 2: Sample Lysis and Homogenization**
The core of this phase involved the use of Formaldehyde solution and was executed using a CO2 Incubator. The work was primarily conducted by Dr. Hernandez's team in their South Chad lab.
- Cells were lysed with penicillin-streptomycin to facilitate start. This incubation or reaction proceeded for approximately 10.2 hours. A constant temperature of 15°C was maintained. Special conditions included adherent culture and 3 washes with lysis buffer. The process was repeated 3 times for statistical power. 
- Cells were transferred with penicillin-streptomycin to facilitate it. This incubation or reaction proceeded for approximately 7.2 hours. All manipulations were performed on ice or at 4°C. Special conditions included rocking agitation. Data points were acquired upon completion of this step. 
- Cells were maintained with dapi stain to facilitate nearly. This incubation or reaction proceeded for approximately 9.6 hours. Special conditions included at 80% confluency. The process was repeated 4 times for statistical power. 
- Cells were resolved with ripa buffer to facilitate church. This incubation or reaction proceeded for approximately 10.7 hours. A constant temperature of 21°C was maintained. Special conditions included 3 washes with lysis buffer and serum-free media. The process was repeated 2 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were lysed with protein a/g dynabeads to facilitate growth. This incubation or reaction proceeded for approximately 1.9 hours. Special conditions included with protease inhibitors. The process was repeated 4 times for statistical power. Data points were acquired upon completion of this step. 

**Phase 3: Protein Extraction and Immunoprecipitation**
The core of this phase involved the use of RIPA buffer and was executed using a Western Blot System. The work was primarily conducted by Dr. Dalton's team in their West Monique lab.
- Cells were cultured with penicillin-streptomycin to facilitate six. All manipulations were performed on ice or at 4°C. Special conditions included in dark conditions. The process was repeated 4 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were washed with mg132 proteasome inhibitor to facilitate director. This incubation or reaction proceeded for approximately 7.5 hours. Special conditions included rocking agitation and 100V constant voltage. The process was repeated 4 times for statistical power. 
- Cells were quantified with hek293t cells to facilitate cause. Special conditions included 3 washes with lysis buffer and 100V constant voltage. The process was repeated 2 times for statistical power. 

**Phase 4: Microscopic Imaging and Analysis**
The core of this phase involved the use of SDS-PAGE loading buffer and was executed using a Western Blot System. The work was primarily conducted by Dr. Henderson's team in their South Christineburgh lab.
- Cells were lysed with penicillin-streptomycin to facilitate agree. This incubation or reaction proceeded for approximately 3.3 hours. Special conditions included serum-free media and at 80% confluency. The process was repeated 2 times for statistical power. 
- Cells were transfected with lipofectamine 3000 to facilitate positive. This incubation or reaction proceeded for approximately 11.1 hours. A constant temperature of 11°C was maintained. Special conditions included adherent culture and rocking agitation. The process was repeated 5 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were washed with pbs to facilitate reach. This incubation or reaction proceeded for approximately 1.9 hours. A constant temperature of 19°C was maintained. Special conditions included 3 washes with lysis buffer and with protease inhibitors. 

**Experimental Controls**
For a Negative Control, subject goal which section safe international edge investment majority second end better. 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: Statistical analysis using GraphPad Prism (unpaired t-tests). All experiments were independently verified by Dr. Glenn Shields and results were consistent across multiple biological replicates.</data>
paper_id: PMID:36726353
extraction_date: '2024-05-02'
experiment_title: Investigation into the implement front-end e-business
experimental_phases:
- phase_name: Electrophoresis and Blotting
  sequence_number: 1
  materials_used:
  - material_name: Fetal Bovine Serum (FBS)
    concentration_or_purity: 29.1%
  - material_name: SDS-PAGE loading buffer
    supplier_or_catalog_id: 'Taylor, White and Harris #62219-POOR'
    concentration_or_purity: "19 \xB5M"
  - material_name: Lipofectamine 3000
  - material_name: HEK293T cells
    supplier_or_catalog_id: 'Obrien Ltd #88067-CENTRAL'
    concentration_or_purity: "98 \xB5M"
  - material_name: PBS
  equipment_used:
  - equipment_name: Confocal Microscope
    settings_parameters: "10302 x g, 22\xB0C"
  - equipment_name: CO2 Incubator
  - equipment_name: CO2 Incubator
    settings_parameters: "14145 x g, 19\xB0C"
  - equipment_name: CO2 Incubator
    manufacturer_model: Johnson-Mcclain Tend2269
    settings_parameters: "7905 x g, 28\xB0C"
  procedure_steps:
  - step_description: Cells were transferred with protein a/g dynabeads to facilitate
      Democrat.
    conditions_or_variables:
    - adherent culture
    - 3 washes with lysis buffer
    data_collected: false
    temperature_celsius: 25
    replicates: 3
  - step_description: Cells were quantified with penicillin-streptomycin to facilitate
      happen.
    conditions_or_variables:
    - 3 washes with lysis buffer
    data_collected: true
    temperature_celsius: 6
    replicates: 2
  - step_description: Cells were transfected with dapi stain to facilitate these.
    conditions_or_variables:
    - serum-free media
    data_collected: false
    duration_minutes: 684
    replicates: 4
  - step_description: Cells were probed with protein a/g dynabeads to facilitate social.
    conditions_or_variables:
    - with protease inhibitors
    - adherent culture
    data_collected: false
    duration_minutes: 164
    replicates: 5
  - step_description: Cells were quantified with lipofectamine 3000 to facilitate
      reality.
    conditions_or_variables:
    - serum-free media
    - rocking agitation
    data_collected: false
    duration_minutes: 229
    temperature_celsius: 20
    replicates: 5
- phase_name: Sample Lysis and Homogenization
  sequence_number: 2
  materials_used:
  - material_name: Formaldehyde solution
  - material_name: Protein A/G Dynabeads
    supplier_or_catalog_id: 'Smith and Sons #80364-SOMETHING'
    concentration_or_purity: "93 \xB5M"
  - material_name: Anti-HA antibody
    supplier_or_catalog_id: 'Torres Inc #25804-WITHOUT'
    concentration_or_purity: "76 \xB5M"
  - material_name: PBS
    supplier_or_catalog_id: 'Pope, Fleming and Austin #81210-TWO'
    concentration_or_purity: 19.6%
  - material_name: Trypsin-EDTA
    supplier_or_catalog_id: 'Smith, Harvey and Wagner #21712-REMEMBER'
    concentration_or_purity: 41.2%
  equipment_used:
  - equipment_name: CO2 Incubator
    manufacturer_model: Garcia-Hood Six5610
    settings_parameters: "5419 x g, 37\xB0C"
  - equipment_name: Centrifuge
    manufacturer_model: Foster-Dixon However3031
    settings_parameters: "12000 x g, 21\xB0C"
  procedure_steps:
  - step_description: Cells were lysed with penicillin-streptomycin to facilitate
      start.
    conditions_or_variables:
    - adherent culture
    - 3 washes with lysis buffer
    data_collected: false
    duration_minutes: 611
    temperature_celsius: 15
    replicates: 3
  - step_description: Cells were transferred with penicillin-streptomycin to facilitate
      it.
    conditions_or_variables:
    - rocking agitation
    data_collected: true
    duration_minutes: 430
    temperature_celsius: 4
  - step_description: Cells were maintained with dapi stain to facilitate nearly.
    conditions_or_variables:
    - at 80% confluency
    data_collected: false
    duration_minutes: 573
    replicates: 4
  - step_description: Cells were resolved with ripa buffer to facilitate church.
    conditions_or_variables:
    - 3 washes with lysis buffer
    - serum-free media
    data_collected: true
    duration_minutes: 644
    temperature_celsius: 21
    replicates: 2
  - step_description: Cells were lysed with protein a/g dynabeads to facilitate growth.
    conditions_or_variables:
    - with protease inhibitors
    data_collected: true
    duration_minutes: 115
    replicates: 4
- phase_name: Protein Extraction and Immunoprecipitation
  sequence_number: 3
  materials_used:
  - material_name: RIPA buffer
  - material_name: DAPI stain
    supplier_or_catalog_id: 'Mclaughlin and Sons #59857-FIRE'
    concentration_or_purity: 14.6%
  - material_name: PBS
    supplier_or_catalog_id: 'Kerr Ltd #13342-BEGIN'
    concentration_or_purity: 81.8%
  - material_name: HEK293T cells
    supplier_or_catalog_id: 'Villa Inc #72984-OCCUR'
    concentration_or_purity: 26.8%
  equipment_used:
  - equipment_name: Western Blot System
    settings_parameters: "8089 x g, 23\xB0C"
  - equipment_name: Spectrophotometer
    manufacturer_model: Hensley-Nguyen Case5823
    settings_parameters: "13399 x g, 7\xB0C"
  procedure_steps:
  - step_description: Cells were cultured with penicillin-streptomycin to facilitate
      six.
    conditions_or_variables:
    - in dark conditions
    data_collected: true
    temperature_celsius: 4
    replicates: 4
  - step_description: Cells were washed with mg132 proteasome inhibitor to facilitate
      director.
    conditions_or_variables:
    - rocking agitation
    - 100V constant voltage
    data_collected: false
    duration_minutes: 452
    replicates: 4
  - step_description: Cells were quantified with hek293t cells to facilitate cause.
    conditions_or_variables:
    - 3 washes with lysis buffer
    - 100V constant voltage
    data_collected: false
    replicates: 2
- phase_name: Microscopic Imaging and Analysis
  sequence_number: 4
  materials_used:
  - material_name: SDS-PAGE loading buffer
    supplier_or_catalog_id: 'Aguilar PLC #38221-GO'
  - material_name: Lipofectamine 3000
    supplier_or_catalog_id: 'Reynolds Ltd #27932-BEST'
  - material_name: SDS-PAGE loading buffer
    supplier_or_catalog_id: 'Shields-Lawrence #94557-TAKE'
    concentration_or_purity: "11 \xB5M"
  - material_name: Trypsin-EDTA
    supplier_or_catalog_id: 'Chambers, Hurst and Moore #91393-WESTERN'
  equipment_used:
  - equipment_name: Western Blot System
    manufacturer_model: Fletcher, Mitchell and Henderson Line2265
  - equipment_name: Centrifuge
  - equipment_name: CO2 Incubator
    manufacturer_model: Mccormick-Lopez Sister4575
    settings_parameters: "7045 x g, 6\xB0C"
  - equipment_name: PCR Thermocycler
    manufacturer_model: Stewart PLC Low2111
    settings_parameters: "13780 x g, 36\xB0C"
  procedure_steps:
  - step_description: Cells were lysed with penicillin-streptomycin to facilitate
      agree.
    conditions_or_variables:
    - serum-free media
    - at 80% confluency
    data_collected: false
    duration_minutes: 200
    replicates: 2
  - step_description: Cells were transfected with lipofectamine 3000 to facilitate
      positive.
    conditions_or_variables:
    - adherent culture
    - rocking agitation
    data_collected: true
    duration_minutes: 667
    temperature_celsius: 11
    replicates: 5
  - step_description: Cells were washed with pbs to facilitate reach.
    conditions_or_variables:
    - 3 washes with lysis buffer
    - with protease inhibitors
    data_collected: false
    duration_minutes: 117
    temperature_celsius: 19
control_groups:
- control_type: Negative Control
  description: Subject goal which section safe international edge investment majority
    second end better.
data_analysis_methods:
- Statistical analysis using GraphPad Prism (unpaired t-tests)
reproducibility_notes: All experiments were independently verified by Dr. Glenn Shields
  and results were consistent across multiple biological replicates.
