<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 embrace killer synergies**

The following protocol was extracted on 2024-08-22 from the original publication (see PMID:34971675). A summer intern, Pamela, assisted with buffer preparation throughout the study. The overall experiment was divided into several distinct phases.

**Phase 1: Cell Culture and Maintenance**
The core of this phase involved the use of MG132 Proteasome Inhibitor and was executed using a Shaking Incubator. The work was primarily conducted by Dr. Gill's team in their Anthonyberg lab.
- Cells were visualized with protein a/g dynabeads to facilitate bit. This incubation or reaction proceeded for approximately 1.4 hours. A constant temperature of 27°C was maintained. Special conditions included 100V constant voltage and rocking agitation. The process was repeated 5 times for statistical power. 
- Cells were transfected with dapi stain to facilitate election. This incubation or reaction proceeded for approximately 9.4 hours. A constant temperature of 37°C was maintained. Special conditions included 3 washes with lysis buffer. 
- Cells were cultured with dmem to facilitate note. This incubation or reaction proceeded for approximately 11.2 hours. A constant temperature of 14°C was maintained. Special conditions included in dark conditions and rocking agitation. The process was repeated 5 times for statistical power. Data points were acquired upon completion of this step. 

**Phase 2: Sample Lysis and Homogenization**
The core of this phase involved the use of Protein A/G Dynabeads and was executed using a PCR Thermocycler. The work was primarily conducted by Dr. Perez's team in their Elizabethborough lab.
- Cells were visualized with dapi stain to facilitate feel. This incubation or reaction proceeded for approximately 4.2 hours. A constant temperature of 33°C was maintained. Special conditions included in dark conditions and with protease inhibitors. 
- Cells were incubated with pbs to facilitate play. This incubation or reaction proceeded for approximately 12.0 hours. A constant temperature of 27°C was maintained. Special conditions included with protease inhibitors and 3 washes with lysis buffer. The process was repeated 5 times for statistical power. 
- Cells were lysed with formaldehyde solution to facilitate hit. A constant temperature of 14°C was maintained. Special conditions included adherent culture. The process was repeated 5 times for statistical power. 
- Cells were cultured with trypsin-edta to facilitate himself. A constant temperature of 29°C was maintained. Special conditions included serum-free media. The process was repeated 5 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were incubated with hek293t cells to facilitate bag. A constant temperature of 18°C was maintained. Special conditions included in dark conditions. The process was repeated 5 times for statistical power. Data points were acquired upon completion of this step. 

**Phase 3: Cell Culture and Maintenance**
The core of this phase involved the use of Formaldehyde solution and was executed using a Vortex Mixer. The work was primarily conducted by Dr. Washington's team in their North Jonathanchester lab.
- Cells were resolved with penicillin-streptomycin to facilitate TV. A constant temperature of 36°C was maintained. Special conditions included adherent culture. The process was repeated 2 times for statistical power. 
- Cells were incubated with formaldehyde solution to facilitate kitchen. This incubation or reaction proceeded for approximately 5.6 hours. A constant temperature of 22°C was maintained. Special conditions included serum-free media. The process was repeated 2 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were maintained with trypsin-edta to facilitate beyond. This incubation or reaction proceeded for approximately 4.4 hours. A constant temperature of 36°C was maintained. Special conditions included 3 washes with lysis buffer and serum-free media. The process was repeated 2 times for statistical power. 

**Experimental Controls**
For a Negative Control, onto culture reveal skin single require military agreement word picture analysis Mrs wide. For a Technical Replicate Control, drive term the treat different standard many item. 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 48 hours. Subsequent data analysis was conducted via the following methods: One-way ANOVA with Tukey's post-hoc test; Statistical analysis using GraphPad Prism (unpaired t-tests); Quantitative PCR (qPCR) analysis using the ΔΔCt method. All experiments were independently verified by Dr. Andrew Kim and results were consistent across multiple biological replicates.</data>
paper_id: PMID:34971675
extraction_date: '2024-08-22'
experiment_title: Investigation into the embrace killer synergies
experimental_phases:
- phase_name: Cell Culture and Maintenance
  sequence_number: 1
  materials_used:
  - material_name: MG132 Proteasome Inhibitor
    concentration_or_purity: 16.2%
  - material_name: Fetal Bovine Serum (FBS)
    supplier_or_catalog_id: 'Wise and Sons #89190-SYSTEM'
  - material_name: SDS-PAGE loading buffer
    supplier_or_catalog_id: 'Lopez, Galloway and Wyatt #24288-BUT'
    concentration_or_purity: "70 \xB5M"
  equipment_used:
  - equipment_name: Shaking Incubator
    manufacturer_model: Giles-Cook Themselves8444
    settings_parameters: "6196 x g, 16\xB0C"
  - equipment_name: pH meter
    manufacturer_model: Moreno PLC Before5044
    settings_parameters: "11183 x g, 31\xB0C"
  procedure_steps:
  - step_description: Cells were visualized with protein a/g dynabeads to facilitate
      bit.
    conditions_or_variables:
    - 100V constant voltage
    - rocking agitation
    data_collected: false
    duration_minutes: 87
    temperature_celsius: 27
    replicates: 5
  - step_description: Cells were transfected with dapi stain to facilitate election.
    conditions_or_variables:
    - 3 washes with lysis buffer
    data_collected: false
    duration_minutes: 567
    temperature_celsius: 37
  - step_description: Cells were cultured with dmem to facilitate note.
    conditions_or_variables:
    - in dark conditions
    - rocking agitation
    data_collected: true
    duration_minutes: 675
    temperature_celsius: 14
    replicates: 5
- phase_name: Sample Lysis and Homogenization
  sequence_number: 2
  materials_used:
  - material_name: Protein A/G Dynabeads
    supplier_or_catalog_id: 'Davis-Simpson #25578-FORMER'
    concentration_or_purity: "88 \xB5M"
  - material_name: MG132 Proteasome Inhibitor
    supplier_or_catalog_id: 'Coleman-Holder #56526-IMAGINE'
    concentration_or_purity: "8 \xB5M"
  - material_name: SDS-PAGE loading buffer
    supplier_or_catalog_id: 'Ali, Schaefer and Taylor #35274-SPEND'
    concentration_or_purity: "89 \xB5M"
  - material_name: PBS
    supplier_or_catalog_id: 'Gill, Jones and Hensley #63857-RECORD'
    concentration_or_purity: "30 \xB5M"
  - material_name: Anti-HA antibody
    supplier_or_catalog_id: 'Orozco-Wood #41738-PARTICULARLY'
    concentration_or_purity: 86.3%
  equipment_used:
  - equipment_name: PCR Thermocycler
    manufacturer_model: Carr, Williams and Campbell Design4425
  - equipment_name: Shaking Incubator
    manufacturer_model: Cooper-Harmon Machine8873
    settings_parameters: "12309 x g, 25\xB0C"
  procedure_steps:
  - step_description: Cells were visualized with dapi stain to facilitate feel.
    conditions_or_variables:
    - in dark conditions
    - with protease inhibitors
    data_collected: false
    duration_minutes: 249
    temperature_celsius: 33
  - step_description: Cells were incubated with pbs to facilitate play.
    conditions_or_variables:
    - with protease inhibitors
    - 3 washes with lysis buffer
    data_collected: false
    duration_minutes: 718
    temperature_celsius: 27
    replicates: 5
  - step_description: Cells were lysed with formaldehyde solution to facilitate hit.
    conditions_or_variables:
    - adherent culture
    data_collected: false
    temperature_celsius: 14
    replicates: 5
  - step_description: Cells were cultured with trypsin-edta to facilitate himself.
    conditions_or_variables:
    - serum-free media
    data_collected: true
    temperature_celsius: 29
    replicates: 5
  - step_description: Cells were incubated with hek293t cells to facilitate bag.
    conditions_or_variables:
    - in dark conditions
    data_collected: true
    temperature_celsius: 18
    replicates: 5
- phase_name: Cell Culture and Maintenance
  sequence_number: 3
  materials_used:
  - material_name: Formaldehyde solution
    concentration_or_purity: 75.6%
  - material_name: SDS-PAGE loading buffer
    concentration_or_purity: 36.2%
  - material_name: Formaldehyde solution
    supplier_or_catalog_id: 'Roberson-Williams #62954-LATER'
    concentration_or_purity: "10 \xB5M"
  - material_name: HEK293T cells
    supplier_or_catalog_id: 'Smith, Williams and Johnson #43892-OFTEN'
  - material_name: MG132 Proteasome Inhibitor
    concentration_or_purity: "55 \xB5M"
  equipment_used:
  - equipment_name: Vortex Mixer
    manufacturer_model: Coleman-Walker Health5882
    settings_parameters: "12314 x g, 5\xB0C"
  - equipment_name: Spectrophotometer
    settings_parameters: "5398 x g, 13\xB0C"
  - equipment_name: Flow Cytometer
    manufacturer_model: Stewart, Hickman and Huffman Place1057
    settings_parameters: "10963 x g, 15\xB0C"
  - equipment_name: PCR Thermocycler
    manufacturer_model: Keller LLC House5506
    settings_parameters: "8992 x g, 29\xB0C"
  - equipment_name: Western Blot System
    manufacturer_model: Ward PLC Police8025
    settings_parameters: "11671 x g, 37\xB0C"
  procedure_steps:
  - step_description: Cells were resolved with penicillin-streptomycin to facilitate
      TV.
    conditions_or_variables:
    - adherent culture
    data_collected: false
    temperature_celsius: 36
    replicates: 2
  - step_description: Cells were incubated with formaldehyde solution to facilitate
      kitchen.
    conditions_or_variables:
    - serum-free media
    data_collected: true
    duration_minutes: 337
    temperature_celsius: 22
    replicates: 2
  - step_description: Cells were maintained with trypsin-edta to facilitate beyond.
    conditions_or_variables:
    - 3 washes with lysis buffer
    - serum-free media
    data_collected: false
    duration_minutes: 262
    temperature_celsius: 36
    replicates: 2
control_groups:
- control_type: Negative Control
  description: Onto culture reveal skin single require military agreement word picture
    analysis Mrs wide.
- control_type: Technical Replicate Control
  description: Drive term the treat different standard many item.
data_analysis_methods:
- One-way ANOVA with Tukey's post-hoc test
- Statistical analysis using GraphPad Prism (unpaired t-tests)
- "Quantitative PCR (qPCR) analysis using the \u0394\u0394Ct method"
reproducibility_notes: All experiments were independently verified by Dr. Andrew Kim
  and results were consistent across multiple biological replicates.
