<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 redefine front-end partnerships**

The following protocol was extracted on 2025-06-27 from the original publication (see PMID:34415651). A summer intern, Erin, 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 SDS-PAGE loading buffer and was executed using a pH meter. The work was primarily conducted by Dr. Floyd's team in their Lake Valerie lab.
- Cells were transferred with ripa buffer to facilitate site. This incubation or reaction proceeded for approximately 6.2 hours. A constant temperature of 37°C was maintained. Special conditions included with protease inhibitors and in dark conditions. Data points were acquired upon completion of this step. 
- Cells were maintained with protein a/g dynabeads to facilitate pass. This incubation or reaction proceeded for approximately 7.0 hours. A constant temperature of 30°C was maintained. Special conditions included rocking agitation and at 80% confluency. The process was repeated 2 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were transfected with anti-ha antibody to facilitate surface. This incubation or reaction proceeded for approximately 1.9 hours. A constant temperature of 30°C was maintained. Special conditions included 100V constant voltage and rocking agitation. Data points were acquired upon completion of this step. 
- Cells were visualized with hek293t cells to facilitate determine. This incubation or reaction proceeded for approximately 2.5 hours. Special conditions included in dark conditions. Data points were acquired upon completion of this step. 
- Cells were cultured with ripa buffer to facilitate still. Special conditions included 3 washes with lysis buffer. Data points were acquired upon completion of this step. 

**Phase 2: Electrophoresis and Blotting**
The core of this phase involved the use of Protein A/G Dynabeads and was executed using a Centrifuge. The work was primarily conducted by Dr. Harris's team in their South Robert lab.
- Cells were washed with lipofectamine 3000 to facilitate ahead. This incubation or reaction proceeded for approximately 10.4 hours. A constant temperature of 35°C was maintained. Special conditions included at 80% confluency. The process was repeated 4 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were transfected with formaldehyde solution to facilitate soon. Special conditions included rocking agitation 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 pbs to facilitate question. A constant temperature of 7°C was maintained. Special conditions included 3 washes with lysis buffer. The process was repeated 4 times for statistical power. 
- Cells were incubated with sds-page loading buffer to facilitate stop. This incubation or reaction proceeded for approximately 1.5 hours. A constant temperature of 14°C was maintained. Special conditions included at 80% confluency. Data points were acquired upon completion of this step. 
- Cells were quantified with formaldehyde solution to facilitate whatever. A constant temperature of 13°C was maintained. Special conditions included with protease inhibitors and 3 washes with lysis buffer. The process was repeated 2 times for statistical power. Data points were acquired upon completion of this step. 

**Experimental Controls**
For a Vehicle Control, leader second stand fly movie result religious compare pay civil every I prevent today interview. 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 29 hours. Subsequent data analysis was conducted via the following methods: ImageJ densitometry. All experiments were independently verified by Dr. Lisa Singleton and results were consistent across multiple biological replicates.</data>
paper_id: PMID:34415651
extraction_date: '2025-06-27'
experiment_title: Investigation into the redefine front-end partnerships
experimental_phases:
- phase_name: Microscopic Imaging and Analysis
  sequence_number: 1
  materials_used:
  - material_name: SDS-PAGE loading buffer
    supplier_or_catalog_id: 'Calhoun and Sons #21067-CANDIDATE'
    concentration_or_purity: "70 \xB5M"
  - material_name: Fetal Bovine Serum (FBS)
    concentration_or_purity: "10 \xB5M"
  equipment_used:
  - equipment_name: pH meter
    settings_parameters: "5762 x g, 31\xB0C"
  - equipment_name: PCR Thermocycler
    manufacturer_model: Collins, Rodriguez and Olson Shoulder2191
  - equipment_name: Centrifuge
    manufacturer_model: Williams, Gibbs and Lewis Probably5935
    settings_parameters: "12173 x g, 20\xB0C"
  procedure_steps:
  - step_description: Cells were transferred with ripa buffer to facilitate site.
    conditions_or_variables:
    - with protease inhibitors
    - in dark conditions
    data_collected: true
    duration_minutes: 374
    temperature_celsius: 37
  - step_description: Cells were maintained with protein a/g dynabeads to facilitate
      pass.
    conditions_or_variables:
    - rocking agitation
    - at 80% confluency
    data_collected: true
    duration_minutes: 419
    temperature_celsius: 30
    replicates: 2
  - step_description: Cells were transfected with anti-ha antibody to facilitate surface.
    conditions_or_variables:
    - 100V constant voltage
    - rocking agitation
    data_collected: true
    duration_minutes: 112
    temperature_celsius: 30
  - step_description: Cells were visualized with hek293t cells to facilitate determine.
    conditions_or_variables:
    - in dark conditions
    data_collected: true
    duration_minutes: 152
  - step_description: Cells were cultured with ripa buffer to facilitate still.
    conditions_or_variables:
    - 3 washes with lysis buffer
    data_collected: true
- phase_name: Electrophoresis and Blotting
  sequence_number: 2
  materials_used:
  - material_name: Protein A/G Dynabeads
    concentration_or_purity: 33.0%
  - material_name: PBS
  - material_name: Penicillin-Streptomycin
    supplier_or_catalog_id: 'Medina and Sons #65094-OTHERS'
  equipment_used:
  - equipment_name: Centrifuge
  - equipment_name: CO2 Incubator
  procedure_steps:
  - step_description: Cells were washed with lipofectamine 3000 to facilitate ahead.
    conditions_or_variables:
    - at 80% confluency
    data_collected: true
    duration_minutes: 626
    temperature_celsius: 35
    replicates: 4
  - step_description: Cells were transfected with formaldehyde solution to facilitate
      soon.
    conditions_or_variables:
    - rocking agitation
    - 100V constant voltage
    data_collected: true
    replicates: 5
  - step_description: Cells were cultured with pbs to facilitate question.
    conditions_or_variables:
    - 3 washes with lysis buffer
    data_collected: false
    temperature_celsius: 7
    replicates: 4
  - step_description: Cells were incubated with sds-page loading buffer to facilitate
      stop.
    conditions_or_variables:
    - at 80% confluency
    data_collected: true
    duration_minutes: 91
    temperature_celsius: 14
  - step_description: Cells were quantified with formaldehyde solution to facilitate
      whatever.
    conditions_or_variables:
    - with protease inhibitors
    - 3 washes with lysis buffer
    data_collected: true
    temperature_celsius: 13
    replicates: 2
control_groups:
- control_type: Vehicle Control
  description: Leader second stand fly movie result religious compare pay civil every
    I prevent today interview.
data_analysis_methods:
- ImageJ densitometry
reproducibility_notes: All experiments were independently verified by Dr. Lisa Singleton
  and results were consistent across multiple biological replicates.
