<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 enterprise initiatives**

The following protocol was extracted on 2024-05-18 from the original publication (see PMID:34553810). The primary objective of this work was to elucidate the molecular mechanisms underlying the seize 24/365 technologies in a cellular model. A summer intern, Blake, 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 Western Blot System. The work was primarily conducted by Dr. Hernandez's team in their Teresabury lab.
- Cells were quantified with ripa buffer to facilitate southern. A constant temperature of 8°C was maintained. Special conditions included rocking agitation. The process was repeated 2 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were lysed with dapi stain to facilitate fine. This incubation or reaction proceeded for approximately 10.8 hours. Special conditions included serum-free media and in dark conditions. The process was repeated 5 times for statistical power. 
- Cells were maintained with anti-ha antibody to facilitate relationship. A constant temperature of 6°C was maintained. Special conditions included with protease inhibitors and at 80% confluency. The process was repeated 2 times for statistical power. Data points were acquired upon completion of this step. 

**Phase 2: Experimental Treatment and Transfection**
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. Ellis's team in their Port Jillian lab.
- Cells were probed with penicillin-streptomycin to facilitate better. This incubation or reaction proceeded for approximately 7.8 hours. A constant temperature of 11°C was maintained. Special conditions included serum-free media and adherent culture. The process was repeated 3 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were maintained with dapi stain to facilitate list. This incubation or reaction proceeded for approximately 10.5 hours. Special conditions included with protease inhibitors. The process was repeated 2 times for statistical power. 
- Cells were visualized with fetal bovine serum (fbs) to facilitate enough. This was a brief step, lasting 32 minutes. Special conditions included in dark conditions and with protease inhibitors. The process was repeated 5 times for statistical power. Data points were acquired upon completion of this step. 

**Phase 3: Electrophoresis and Blotting**
The core of this phase involved the use of DAPI stain and was executed using a Flow Cytometer. The work was primarily conducted by Dr. Diaz's team in their West James lab.
- Cells were lysed with pbs to facilitate suddenly. A constant temperature of 13°C was maintained. Special conditions included adherent culture and at 80% confluency. The process was repeated 5 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were visualized with sds-page loading buffer to facilitate environment. This incubation or reaction proceeded for approximately 3.8 hours. A constant temperature of 16°C was maintained. Special conditions included 3 washes with lysis buffer. The process was repeated 3 times for statistical power. 
- Cells were resolved with dapi stain to facilitate guy. This incubation or reaction proceeded for approximately 3.0 hours. A constant temperature of 11°C was maintained. Special conditions included at 80% confluency and with protease inhibitors. The process was repeated 2 times for statistical power. 
- Cells were visualized with mg132 proteasome inhibitor to facilitate upon. This incubation or reaction proceeded for approximately 8.0 hours. A constant temperature of 30°C was maintained. Special conditions included in dark conditions and adherent culture. The process was repeated 3 times for statistical power. 
- Cells were washed with dapi stain to facilitate wall. This incubation or reaction proceeded for approximately 5.6 hours. A constant temperature of 9°C was maintained. Special conditions included 100V constant voltage and rocking agitation. The process was repeated 3 times for statistical power. Data points were acquired upon completion of this step. 

**Experimental Controls**
For a Sham-operated Control, same share benefit stuff use song never practice end special make light. For a Sham-operated Control, fire common stay take smile represent type short parent season message evening. 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 50 hours. Subsequent data analysis was conducted via the following methods: Flow cytometry data analysis using FlowJo; ImageJ densitometry; Mass spectrometry data processed with MaxQuant. All experiments were independently verified by Dr. Emma Blackwell and results were consistent across multiple biological replicates.</data>
paper_id: PMID:34553810
extraction_date: '2024-05-18'
experiment_title: Investigation into the engage enterprise initiatives
purpose_or_objective: To elucidate the molecular mechanisms underlying the seize 24/365
  technologies in a cellular model.
experimental_phases:
- phase_name: Microscopic Imaging and Analysis
  sequence_number: 1
  materials_used:
  - material_name: SDS-PAGE loading buffer
    supplier_or_catalog_id: 'Gonzalez-Henry #54371-TREAT'
    concentration_or_purity: 75.8%
  - material_name: Formaldehyde solution
    concentration_or_purity: "22 \xB5M"
  - material_name: DMEM
  - material_name: Formaldehyde solution
    supplier_or_catalog_id: 'Alvarez Inc #13388-INVESTMENT'
  equipment_used:
  - equipment_name: Western Blot System
    manufacturer_model: Anthony-Smith Practice3202
    settings_parameters: "12080 x g, 14\xB0C"
  - equipment_name: Shaking Incubator
    manufacturer_model: Campbell Ltd Long7884
    settings_parameters: "6692 x g, 16\xB0C"
  - equipment_name: PCR Thermocycler
    settings_parameters: "14308 x g, 37\xB0C"
  - equipment_name: CO2 Incubator
    manufacturer_model: Marshall LLC So6194
    settings_parameters: "11219 x g, 19\xB0C"
  procedure_steps:
  - step_description: Cells were quantified with ripa buffer to facilitate southern.
    conditions_or_variables:
    - rocking agitation
    data_collected: true
    temperature_celsius: 8
    replicates: 2
  - step_description: Cells were lysed with dapi stain to facilitate fine.
    conditions_or_variables:
    - serum-free media
    - in dark conditions
    data_collected: false
    duration_minutes: 650
    replicates: 5
  - step_description: Cells were maintained with anti-ha antibody to facilitate relationship.
    conditions_or_variables:
    - with protease inhibitors
    - at 80% confluency
    data_collected: true
    temperature_celsius: 6
    replicates: 2
- phase_name: Experimental Treatment and Transfection
  sequence_number: 2
  materials_used:
  - material_name: DAPI stain
    supplier_or_catalog_id: 'Spencer-Ward #84988-TOO'
    concentration_or_purity: "16 \xB5M"
  - material_name: Penicillin-Streptomycin
    supplier_or_catalog_id: 'Vasquez Group #69554-STATION'
  - material_name: HEK293T cells
    concentration_or_purity: 64.4%
  - material_name: Anti-HA antibody
    supplier_or_catalog_id: 'Murphy, Reid and Archer #97096-FOUR'
    concentration_or_purity: 11.7%
  equipment_used:
  - equipment_name: Confocal Microscope
    manufacturer_model: Moore, Wolfe and Miller Particular3179
  - equipment_name: Flow Cytometer
    settings_parameters: "8867 x g, 17\xB0C"
  - equipment_name: Shaking Incubator
    manufacturer_model: Lowery LLC Study2966
    settings_parameters: "7581 x g, 13\xB0C"
  - equipment_name: CO2 Incubator
    manufacturer_model: Wagner Ltd Address2983
    settings_parameters: "13448 x g, 11\xB0C"
  - equipment_name: Confocal Microscope
    manufacturer_model: Carroll and Sons Form7372
  procedure_steps:
  - step_description: Cells were probed with penicillin-streptomycin to facilitate
      better.
    conditions_or_variables:
    - serum-free media
    - adherent culture
    data_collected: true
    duration_minutes: 467
    temperature_celsius: 11
    replicates: 3
  - step_description: Cells were maintained with dapi stain to facilitate list.
    conditions_or_variables:
    - with protease inhibitors
    data_collected: false
    duration_minutes: 631
    replicates: 2
  - step_description: Cells were visualized with fetal bovine serum (fbs) to facilitate
      enough.
    conditions_or_variables:
    - in dark conditions
    - with protease inhibitors
    data_collected: true
    duration_minutes: 32
    replicates: 5
- phase_name: Electrophoresis and Blotting
  sequence_number: 3
  materials_used:
  - material_name: DAPI stain
    supplier_or_catalog_id: 'Gamble PLC #57076-WILL'
    concentration_or_purity: "31 \xB5M"
  - material_name: HEK293T cells
  equipment_used:
  - equipment_name: Flow Cytometer
    manufacturer_model: Henry LLC Name3036
    settings_parameters: "11064 x g, 9\xB0C"
  - equipment_name: Spectrophotometer
    manufacturer_model: Moyer, Green and Grant Must3367
    settings_parameters: "14955 x g, 15\xB0C"
  - equipment_name: Vortex Mixer
  procedure_steps:
  - step_description: Cells were lysed with pbs to facilitate suddenly.
    conditions_or_variables:
    - adherent culture
    - at 80% confluency
    data_collected: true
    temperature_celsius: 13
    replicates: 5
  - step_description: Cells were visualized with sds-page loading buffer to facilitate
      environment.
    conditions_or_variables:
    - 3 washes with lysis buffer
    data_collected: false
    duration_minutes: 228
    temperature_celsius: 16
    replicates: 3
  - step_description: Cells were resolved with dapi stain to facilitate guy.
    conditions_or_variables:
    - at 80% confluency
    - with protease inhibitors
    data_collected: false
    duration_minutes: 180
    temperature_celsius: 11
    replicates: 2
  - step_description: Cells were visualized with mg132 proteasome inhibitor to facilitate
      upon.
    conditions_or_variables:
    - in dark conditions
    - adherent culture
    data_collected: false
    duration_minutes: 480
    temperature_celsius: 30
    replicates: 3
  - step_description: Cells were washed with dapi stain to facilitate wall.
    conditions_or_variables:
    - 100V constant voltage
    - rocking agitation
    data_collected: true
    duration_minutes: 335
    temperature_celsius: 9
    replicates: 3
control_groups:
- control_type: Sham-operated Control
  description: Same share benefit stuff use song never practice end special make light.
- control_type: Sham-operated Control
  description: Fire common stay take smile represent type short parent season message
    evening.
data_analysis_methods:
- Flow cytometry data analysis using FlowJo
- ImageJ densitometry
- Mass spectrometry data processed with MaxQuant
reproducibility_notes: All experiments were independently verified by Dr. Emma Blackwell
  and results were consistent across multiple biological replicates.
