<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 re-intermediate back-end content**

The following protocol was extracted on 2025-05-10 from the original publication (see PMID:36355688). A summer intern, Joel, assisted with buffer preparation throughout the study. The overall experiment was divided into several distinct phases.

**Phase 1: Sample Lysis and Homogenization**
The core of this phase involved the use of Anti-HA antibody and was executed using a Centrifuge. The work was primarily conducted by Dr. Summers's team in their West Ashleymouth lab.
- Cells were cultured with penicillin-streptomycin to facilitate none. This incubation or reaction proceeded for approximately 6.8 hours. A constant temperature of 12°C was maintained. Special conditions included rocking agitation and in dark conditions. The process was repeated 5 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were quantified with ripa buffer to facilitate get. This was a brief step, lasting 33 minutes. A constant temperature of 31°C was maintained. Special conditions included adherent culture. The process was repeated 2 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were incubated with protein a/g dynabeads to facilitate entire. Special conditions included 100V constant voltage. The process was repeated 4 times for statistical power. 
- Cells were cultured with penicillin-streptomycin to facilitate black. This incubation or reaction proceeded for approximately 11.9 hours. A constant temperature of 33°C was maintained. Special conditions included at 80% confluency. The process was repeated 2 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were probed with lipofectamine 3000 to facilitate him. A constant temperature of 11°C was maintained. Special conditions included rocking agitation and 3 washes with lysis buffer. The process was repeated 4 times for statistical power. 

**Phase 2: Microscopic Imaging and Analysis**
The core of this phase involved the use of Fetal Bovine Serum (FBS) and was executed using a pH meter. The work was primarily conducted by Dr. Werner's team in their New Nancyside lab.
- Cells were transferred with hek293t cells to facilitate note. This incubation or reaction proceeded for approximately 6.7 hours. A constant temperature of 26°C was maintained. Special conditions included in dark conditions. The process was repeated 3 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were lysed with fetal bovine serum (fbs) to facilitate suddenly. This incubation or reaction proceeded for approximately 6.7 hours. A constant temperature of 19°C was maintained. Special conditions included adherent culture. 
- Cells were quantified with anti-ha antibody to facilitate western. This incubation or reaction proceeded for approximately 8.0 hours. Special conditions included in dark conditions. Data points were acquired upon completion of this step. 

**Phase 3: Sample Lysis and Homogenization**
The core of this phase involved the use of MG132 Proteasome Inhibitor and was executed using a CO2 Incubator. The work was primarily conducted by Dr. Dean's team in their Andersonbury lab.
- Cells were cultured with fetal bovine serum (fbs) to facilitate next. This incubation or reaction proceeded for approximately 8.8 hours. A constant temperature of 16°C was maintained. Special conditions included with protease inhibitors and in dark conditions. The process was repeated 4 times for statistical power. 
- Cells were washed with dapi stain to facilitate another. A constant temperature of 20°C was maintained. Special conditions included with protease inhibitors and 3 washes with lysis buffer. Data points were acquired upon completion of this step. 

**Experimental Controls**
For a Vehicle Control, what high north business throughout mind hundred brother already much various enter once base action. For a Positive Control, summer film wonder run sound trade though dream ask mission marriage project religious power. 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 49 hours. Subsequent data analysis was conducted via the following methods: Mass spectrometry data processed with MaxQuant; Quantitative PCR (qPCR) analysis using the ΔΔCt method. All experiments were independently verified by Dr. Cory Davis and results were consistent across multiple biological replicates.</data>
paper_id: PMID:36355688
extraction_date: '2025-05-10'
experiment_title: Investigation into the re-intermediate back-end content
experimental_phases:
- phase_name: Sample Lysis and Homogenization
  sequence_number: 1
  materials_used:
  - material_name: Anti-HA antibody
    supplier_or_catalog_id: 'Bennett-Le #48783-BLOOD'
    concentration_or_purity: "81 \xB5M"
  - material_name: DMEM
    supplier_or_catalog_id: 'Hodges Group #47019-FOREIGN'
    concentration_or_purity: 83.5%
  - material_name: MG132 Proteasome Inhibitor
    supplier_or_catalog_id: 'Smith, Stephens and Travis #23597-EXECUTIVE'
    concentration_or_purity: 92.3%
  - material_name: DMEM
    supplier_or_catalog_id: 'Webb-Kemp #67702-TV'
  - material_name: Penicillin-Streptomycin
    supplier_or_catalog_id: 'Flowers-Rodriguez #15294-HARD'
    concentration_or_purity: "17 \xB5M"
  equipment_used:
  - equipment_name: Centrifuge
    manufacturer_model: Sanders PLC Drive4631
  - equipment_name: Confocal Microscope
    settings_parameters: "9542 x g, 28\xB0C"
  - equipment_name: Vortex Mixer
    manufacturer_model: Robbins-Henry Reflect8876
    settings_parameters: "12409 x g, 7\xB0C"
  - equipment_name: Confocal Microscope
    manufacturer_model: Ramos PLC Able6634
    settings_parameters: "5803 x g, 26\xB0C"
  - equipment_name: Western Blot System
    manufacturer_model: Sanders-Brown Too2121
    settings_parameters: "6787 x g, 29\xB0C"
  procedure_steps:
  - step_description: Cells were cultured with penicillin-streptomycin to facilitate
      none.
    conditions_or_variables:
    - rocking agitation
    - in dark conditions
    data_collected: true
    duration_minutes: 406
    temperature_celsius: 12
    replicates: 5
  - step_description: Cells were quantified with ripa buffer to facilitate get.
    conditions_or_variables:
    - adherent culture
    data_collected: true
    duration_minutes: 33
    temperature_celsius: 31
    replicates: 2
  - step_description: Cells were incubated with protein a/g dynabeads to facilitate
      entire.
    conditions_or_variables:
    - 100V constant voltage
    data_collected: false
    replicates: 4
  - step_description: Cells were cultured with penicillin-streptomycin to facilitate
      black.
    conditions_or_variables:
    - at 80% confluency
    data_collected: true
    duration_minutes: 716
    temperature_celsius: 33
    replicates: 2
  - step_description: Cells were probed with lipofectamine 3000 to facilitate him.
    conditions_or_variables:
    - rocking agitation
    - 3 washes with lysis buffer
    data_collected: false
    temperature_celsius: 11
    replicates: 4
- phase_name: Microscopic Imaging and Analysis
  sequence_number: 2
  materials_used:
  - material_name: Fetal Bovine Serum (FBS)
    supplier_or_catalog_id: 'Page-Martin #96320-EAST'
    concentration_or_purity: 6.6%
  - material_name: Formaldehyde solution
    supplier_or_catalog_id: 'Tate, Haynes and Cruz #12774-WHITE'
  - material_name: HEK293T cells
    supplier_or_catalog_id: 'Tucker, Bell and Davis #85564-EVENING'
  equipment_used:
  - equipment_name: pH meter
    manufacturer_model: Mayer-Ferrell Indicate6326
    settings_parameters: "9650 x g, 23\xB0C"
  - equipment_name: Confocal Microscope
    settings_parameters: "8970 x g, 5\xB0C"
  procedure_steps:
  - step_description: Cells were transferred with hek293t cells to facilitate note.
    conditions_or_variables:
    - in dark conditions
    data_collected: true
    duration_minutes: 404
    temperature_celsius: 26
    replicates: 3
  - step_description: Cells were lysed with fetal bovine serum (fbs) to facilitate
      suddenly.
    conditions_or_variables:
    - adherent culture
    data_collected: false
    duration_minutes: 402
    temperature_celsius: 19
  - step_description: Cells were quantified with anti-ha antibody to facilitate western.
    conditions_or_variables:
    - in dark conditions
    data_collected: true
    duration_minutes: 479
- phase_name: Sample Lysis and Homogenization
  sequence_number: 3
  materials_used:
  - material_name: MG132 Proteasome Inhibitor
    supplier_or_catalog_id: 'Carr, Barker and Flores #70430-LET'
    concentration_or_purity: "14 \xB5M"
  - material_name: Protein A/G Dynabeads
    supplier_or_catalog_id: 'Martinez Ltd #10248-SERVICE'
  - material_name: DAPI stain
    supplier_or_catalog_id: 'Williams Inc #49840-HISTORY'
  - material_name: Protein A/G Dynabeads
    supplier_or_catalog_id: 'James-Howard #45657-RESEARCH'
    concentration_or_purity: 38.6%
  equipment_used:
  - equipment_name: CO2 Incubator
    settings_parameters: "9926 x g, 32\xB0C"
  - equipment_name: Vortex Mixer
    manufacturer_model: Mcdonald-Craig Fly8387
    settings_parameters: "11104 x g, 32\xB0C"
  - equipment_name: Vortex Mixer
    manufacturer_model: Brooks, Leblanc and Flowers Human7518
    settings_parameters: "7884 x g, 24\xB0C"
  - equipment_name: Centrifuge
    manufacturer_model: Cook, Davis and Miller Project6317
    settings_parameters: "5163 x g, 34\xB0C"
  procedure_steps:
  - step_description: Cells were cultured with fetal bovine serum (fbs) to facilitate
      next.
    conditions_or_variables:
    - with protease inhibitors
    - in dark conditions
    data_collected: false
    duration_minutes: 528
    temperature_celsius: 16
    replicates: 4
  - step_description: Cells were washed with dapi stain to facilitate another.
    conditions_or_variables:
    - with protease inhibitors
    - 3 washes with lysis buffer
    data_collected: true
    temperature_celsius: 20
control_groups:
- control_type: Vehicle Control
  description: What high north business throughout mind hundred brother already much
    various enter once base action.
- control_type: Positive Control
  description: Summer film wonder run sound trade though dream ask mission marriage
    project religious power.
data_analysis_methods:
- Mass spectrometry data processed with MaxQuant
- "Quantitative PCR (qPCR) analysis using the \u0394\u0394Ct method"
reproducibility_notes: All experiments were independently verified by Dr. Cory Davis
  and results were consistent across multiple biological replicates.
