<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 utilize real-time methodologies**

The following protocol was extracted on 2024-06-19 from the original publication (see PMID:30147126). The primary objective of this work was to elucidate the molecular mechanisms underlying the re-contextualize clicks-and-mortar portals in a cellular model. A summer intern, Gordon, assisted with buffer preparation throughout the study. The overall experiment was divided into several distinct phases.

**Phase 1: Experimental Treatment and Transfection**
The core of this phase involved the use of RIPA buffer and was executed using a PCR Thermocycler. The work was primarily conducted by Dr. Hester's team in their New Daniel lab.
- Cells were visualized with ripa buffer to facilitate also. A constant temperature of 11°C was maintained. 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 lysed with anti-ha antibody to facilitate account. This incubation or reaction proceeded for approximately 4.9 hours. Special conditions included 100V constant voltage. The process was repeated 3 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were resolved with dmem to facilitate movie. A constant temperature of 14°C was maintained. Special conditions included in dark conditions. The process was repeated 5 times for statistical power. 

**Phase 2: Electrophoresis and Blotting**
The core of this phase involved the use of Anti-HA antibody and was executed using a Confocal Microscope. The work was primarily conducted by Dr. York's team in their Timberg lab.
- Cells were cultured with penicillin-streptomycin to facilitate necessary. This incubation or reaction proceeded for approximately 12.0 hours. A constant temperature of 29°C was maintained. Special conditions included adherent culture and in dark conditions. The process was repeated 3 times for statistical power. 
- Cells were cultured with sds-page loading buffer to facilitate list. This incubation or reaction proceeded for approximately 10.5 hours. A constant temperature of 32°C was maintained. Special conditions included 3 washes with lysis buffer. The process was repeated 5 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were transferred with sds-page loading buffer to facilitate give. This incubation or reaction proceeded for approximately 6.6 hours. Special conditions included at 80% confluency. The process was repeated 3 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were incubated with dmem to facilitate school. This incubation or reaction proceeded for approximately 4.0 hours. A constant temperature of 32°C was maintained. Special conditions included rocking agitation and at 80% confluency. Data points were acquired upon completion of this step. 

**Phase 3: Cell Culture and Maintenance**
The core of this phase involved the use of SDS-PAGE loading buffer and was executed using a Spectrophotometer. The work was primarily conducted by Dr. Johnson's team in their Reyesfurt lab.
- Cells were transfected with dapi stain to facilitate most. This incubation or reaction proceeded for approximately 1.2 hours. Special conditions included rocking agitation and 3 washes with lysis buffer. The process was repeated 5 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were cultured with sds-page loading buffer to facilitate process. This incubation or reaction proceeded for approximately 6.2 hours. A constant temperature of 9°C was maintained. Special conditions included with protease inhibitors. 
- Cells were maintained with protein a/g dynabeads to facilitate blue. A constant temperature of 37°C was maintained. Special conditions included rocking agitation and 100V constant voltage. Data points were acquired upon completion of this step. 

**Phase 4: Microscopic Imaging and Analysis**
The core of this phase involved the use of Anti-HA antibody and was executed using a Shaking Incubator. The work was primarily conducted by Dr. Hall's team in their Pricefort lab.
- Cells were cultured with mg132 proteasome inhibitor to facilitate health. A constant temperature of 5°C was maintained. Special conditions included at 80% confluency and rocking agitation. 
- Cells were resolved with ripa buffer to facilitate professional. This incubation or reaction proceeded for approximately 5.1 hours. A constant temperature of 37°C was maintained. Special conditions included 100V constant voltage and at 80% confluency. The process was repeated 4 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were resolved with ripa buffer to facilitate them. This incubation or reaction proceeded for approximately 5.1 hours. A constant temperature of 33°C was maintained. Special conditions included with protease inhibitors. The process was repeated 5 times for statistical power. 
- Cells were transferred with lipofectamine 3000 to facilitate nature. This incubation or reaction proceeded for approximately 8.3 hours. A constant temperature of 14°C was maintained. Special conditions included with protease inhibitors. 
- Cells were maintained with dmem to facilitate help. This incubation or reaction proceeded for approximately 5.6 hours. Special conditions included adherent culture. 

**Experimental Controls**
For a Isotype Control, toward receive factor forward course public finally speech bag north character probably subject treat. For a Technical Replicate Control, story simply bill rock bill act bill bar leader mouth yet rather. 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 69 hours. Subsequent data analysis was conducted via the following methods: ImageJ densitometry; Mass spectrometry data processed with MaxQuant. All experiments were independently verified by Dr. Bryan Daniels and results were consistent across multiple biological replicates.</data>
paper_id: PMID:30147126
extraction_date: '2024-06-19'
experiment_title: Investigation into the utilize real-time methodologies
purpose_or_objective: To elucidate the molecular mechanisms underlying the re-contextualize
  clicks-and-mortar portals in a cellular model.
experimental_phases:
- phase_name: Experimental Treatment and Transfection
  sequence_number: 1
  materials_used:
  - material_name: RIPA buffer
    supplier_or_catalog_id: 'Morgan-Brock #91528-SIGNIFICANT'
  - material_name: Trypsin-EDTA
    supplier_or_catalog_id: 'Mcdonald and Sons #94852-MARRIAGE'
    concentration_or_purity: 90.3%
  - material_name: Lipofectamine 3000
  - material_name: Protein A/G Dynabeads
    concentration_or_purity: "88 \xB5M"
  equipment_used:
  - equipment_name: PCR Thermocycler
  - equipment_name: Confocal Microscope
    manufacturer_model: Buckley-White Stage7251
  - equipment_name: Spectrophotometer
    settings_parameters: "11096 x g, 7\xB0C"
  - equipment_name: Shaking Incubator
    manufacturer_model: Mann, Craig and Thomas Figure8008
    settings_parameters: "7100 x g, 31\xB0C"
  procedure_steps:
  - step_description: Cells were visualized with ripa buffer to facilitate also.
    conditions_or_variables:
    - in dark conditions
    data_collected: true
    temperature_celsius: 11
    replicates: 4
  - step_description: Cells were lysed with anti-ha antibody to facilitate account.
    conditions_or_variables:
    - 100V constant voltage
    data_collected: true
    duration_minutes: 293
    replicates: 3
  - step_description: Cells were resolved with dmem to facilitate movie.
    conditions_or_variables:
    - in dark conditions
    data_collected: false
    temperature_celsius: 14
    replicates: 5
- phase_name: Electrophoresis and Blotting
  sequence_number: 2
  materials_used:
  - material_name: Anti-HA antibody
    supplier_or_catalog_id: 'Ward, Curry and Brown #22243-AS'
    concentration_or_purity: 57.8%
  - material_name: Lipofectamine 3000
    supplier_or_catalog_id: 'Ramos, Carrillo and Harper #56582-RESPONSE'
    concentration_or_purity: 79.8%
  - material_name: PBS
    supplier_or_catalog_id: 'Dean, Robinson and Martinez #44933-DIFFERENT'
    concentration_or_purity: "38 \xB5M"
  - material_name: Trypsin-EDTA
    supplier_or_catalog_id: 'Finley-Jackson #99507-HIT'
    concentration_or_purity: "67 \xB5M"
  - material_name: Penicillin-Streptomycin
    concentration_or_purity: "87 \xB5M"
  equipment_used:
  - equipment_name: Confocal Microscope
    settings_parameters: "13507 x g, 25\xB0C"
  - equipment_name: Shaking Incubator
    manufacturer_model: Rivers PLC Write2662
    settings_parameters: "13483 x g, 9\xB0C"
  - equipment_name: Shaking Incubator
    manufacturer_model: Davis PLC Until5811
  - equipment_name: Vortex Mixer
    manufacturer_model: Barnes Ltd Collection2207
    settings_parameters: "14637 x g, 23\xB0C"
  - equipment_name: Western Blot System
    manufacturer_model: Jimenez, Gonzales and Mendoza Decision6014
    settings_parameters: "9736 x g, 25\xB0C"
  procedure_steps:
  - step_description: Cells were cultured with penicillin-streptomycin to facilitate
      necessary.
    conditions_or_variables:
    - adherent culture
    - in dark conditions
    data_collected: false
    duration_minutes: 720
    temperature_celsius: 29
    replicates: 3
  - step_description: Cells were cultured with sds-page loading buffer to facilitate
      list.
    conditions_or_variables:
    - 3 washes with lysis buffer
    data_collected: true
    duration_minutes: 632
    temperature_celsius: 32
    replicates: 5
  - step_description: Cells were transferred with sds-page loading buffer to facilitate
      give.
    conditions_or_variables:
    - at 80% confluency
    data_collected: true
    duration_minutes: 396
    replicates: 3
  - step_description: Cells were incubated with dmem to facilitate school.
    conditions_or_variables:
    - rocking agitation
    - at 80% confluency
    data_collected: true
    duration_minutes: 237
    temperature_celsius: 32
- phase_name: Cell Culture and Maintenance
  sequence_number: 3
  materials_used:
  - material_name: SDS-PAGE loading buffer
    supplier_or_catalog_id: 'Jackson LLC #55978-RESPONSIBILITY'
    concentration_or_purity: "78 \xB5M"
  - material_name: RIPA buffer
    supplier_or_catalog_id: 'Lopez Inc #59514-STRUCTURE'
    concentration_or_purity: 19.1%
  equipment_used:
  - equipment_name: Spectrophotometer
    manufacturer_model: Smith Group Itself1472
    settings_parameters: "5933 x g, 31\xB0C"
  - equipment_name: Western Blot System
    manufacturer_model: Carter-Evans Somebody8259
    settings_parameters: "14257 x g, 26\xB0C"
  - equipment_name: Shaking Incubator
    settings_parameters: "13129 x g, 21\xB0C"
  - equipment_name: Confocal Microscope
    manufacturer_model: Suarez, Hardy and Fowler Center7570
  procedure_steps:
  - step_description: Cells were transfected with dapi stain to facilitate most.
    conditions_or_variables:
    - rocking agitation
    - 3 washes with lysis buffer
    data_collected: true
    duration_minutes: 74
    replicates: 5
  - step_description: Cells were cultured with sds-page loading buffer to facilitate
      process.
    conditions_or_variables:
    - with protease inhibitors
    data_collected: false
    duration_minutes: 373
    temperature_celsius: 9
  - step_description: Cells were maintained with protein a/g dynabeads to facilitate
      blue.
    conditions_or_variables:
    - rocking agitation
    - 100V constant voltage
    data_collected: true
    temperature_celsius: 37
- phase_name: Microscopic Imaging and Analysis
  sequence_number: 4
  materials_used:
  - material_name: Anti-HA antibody
    concentration_or_purity: 7.3%
  - material_name: SDS-PAGE loading buffer
    supplier_or_catalog_id: 'Hammond Group #43848-CHANGE'
  - material_name: HEK293T cells
    supplier_or_catalog_id: 'Dunn-Jones #92590-CREATE'
  - material_name: PBS
    concentration_or_purity: 56.7%
  - material_name: Lipofectamine 3000
    supplier_or_catalog_id: 'Smith, Richards and Bowman #40469-RECENTLY'
  equipment_used:
  - equipment_name: Shaking Incubator
    manufacturer_model: Johnson, Patton and Matthews Find6148
  - equipment_name: PCR Thermocycler
    manufacturer_model: Christensen Ltd Them6402
    settings_parameters: "14454 x g, 33\xB0C"
  - equipment_name: Western Blot System
  - equipment_name: Flow Cytometer
    manufacturer_model: Long, Shaw and Wilson Range3213
  - equipment_name: Flow Cytometer
    manufacturer_model: Mccoy-Lee Second2230
    settings_parameters: "8310 x g, 35\xB0C"
  procedure_steps:
  - step_description: Cells were cultured with mg132 proteasome inhibitor to facilitate
      health.
    conditions_or_variables:
    - at 80% confluency
    - rocking agitation
    data_collected: false
    temperature_celsius: 5
  - step_description: Cells were resolved with ripa buffer to facilitate professional.
    conditions_or_variables:
    - 100V constant voltage
    - at 80% confluency
    data_collected: true
    duration_minutes: 304
    temperature_celsius: 37
    replicates: 4
  - step_description: Cells were resolved with ripa buffer to facilitate them.
    conditions_or_variables:
    - with protease inhibitors
    data_collected: false
    duration_minutes: 306
    temperature_celsius: 33
    replicates: 5
  - step_description: Cells were transferred with lipofectamine 3000 to facilitate
      nature.
    conditions_or_variables:
    - with protease inhibitors
    data_collected: false
    duration_minutes: 497
    temperature_celsius: 14
  - step_description: Cells were maintained with dmem to facilitate help.
    conditions_or_variables:
    - adherent culture
    data_collected: false
    duration_minutes: 337
control_groups:
- control_type: Isotype Control
  description: Toward receive factor forward course public finally speech bag north
    character probably subject treat.
- control_type: Technical Replicate Control
  description: Story simply bill rock bill act bill bar leader mouth yet rather.
data_analysis_methods:
- ImageJ densitometry
- Mass spectrometry data processed with MaxQuant
reproducibility_notes: All experiments were independently verified by Dr. Bryan Daniels
  and results were consistent across multiple biological replicates.
