Metabolic Labeling

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Metabolic Labeling

• Rachel Graham
• IGP Methodology
• October 25, 2005
• rachel.graham_at_vanderbilt.edu
• D6221 MCN, 3-0547

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Outline of the Lecture

• What is metabolic labeling? Intro and
  Applications
• Radioisotopic Labeling Advantages and
  Disadvantages
• Radioisotope Methods
• Proteins
• Nucleic Acids
• Nonradioisotope Methods
• BrdU and BrU incorporation
• SILAC labeling
• Problem Solving

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What is Metabolic Labeling?
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What is Metabolic Labeling?

• Takes advantage of the cells own biosynthetic
  processes to introduce detectable markers in
  newly-synthesized molecules
• DNA Replication
• Transcription
• Translation
• Post-translational Modification

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Applications

• Protein synthesis detection and kinetics of
  production
• Protein processing
• Addition of post-translational modifications
• DNA replication and cell proliferation
• RNA synthesis
• Viral replication

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Radioisotopic Labeling Advantages

• Incorporation can be customized
• Defined molecule
• Defined level of radioactivity per molecule
• Easily detectable
• Flexible readout assays
• Quantitative

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Radioisotopic Labeling Disadvantages

• Special precautions required for working with
  radioactivity
• Emission can induce cellular damage and artifacts
  
• Isotope has a window of use
• Usually a trade-off between half-life and
  specific activity

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Radioisotope Methods Protein Detection
Culture Cells
Stimulate Induce Transfect Infect
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Radioisotope Methods Protein Detection
Starve Cells
Culture Cells
Culture without specific amino acids
Stimulate Induce Transfect Infect
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Radioisotope Methods Protein Detection
Starve Cells
Culture Cells
Label Cells
Culture without specific amino acids
Supplement deficient media with radioactive amino
acids
Stimulate Induce Transfect Infect
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Radioisotope Methods Protein Detection
Starve Cells
Culture Cells
Label Cells
Culture without specific amino acids
Harvest Cells
Supplement deficient media with radioactive amino
acids
Stimulate Induce Transfect Infect
13
Radioisotope Methods Protein Detection
Starve Cells
Culture Cells
Label Cells
Culture without specific amino acids
Harvest Cells
Supplement deficient media with radioactive amino
acids
Stimulate Induce Transfect Infect
Detection
Immunoprecipitation
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Radioisotope Methods Reagents for Protein
Labeling

• Complete medium
• Medium lacking amino acids (e.g., -Met/-Cys)
• Radiolabeled amino acids (e.g., 35S Met/Cys)
• Cell lysis buffer
• Assay-dependent
• Actinomycin D (complexes with DNA and inhibits
  cellular DNA-dependent RNA synthesis)
• Cycloheximide (inhibits translation by inhibiting
  peptidyl transferase activity of the ribosome)

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Radioisotope Methods Pulse-Label

• Used to detect protein produced in a given amount
  of time (the pulse)
• 10 minutes to several hours
• Short pulse detects rapidly translated proteins
• Long pulse detects proteins with long turnover
  rates
• Time course of pulses detects protein production
  over time

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Radioisotope Methods Pulse Label Basic Protocol
Culture Cells
Stimulate Induce Transfect Infect
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Radioisotope Methods Pulse Label Basic Protocol
Starve Cells
Culture Cells
Culture without specific amino acids (ActD)
Stimulate Induce Transfect Infect
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Radioisotope Methods Pulse Label Basic Protocol
Starve Cells
Culture Cells
Label Cells
Culture without specific amino acids (ActD)
Supplement deficient media with radioactive amino
acids
Stimulate Induce Transfect Infect
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Radioisotope Methods Pulse Label Basic Protocol
Starve Cells
Culture Cells
Label Cells
Culture without specific amino acids (ActD)
Harvest Cells
Supplement deficient media with radioactive amino
acids
Stimulate Induce Transfect Infect
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Radioisotope Methods Pulse Label Basic Protocol
Starve Cells
Culture Cells
Label Cells
Culture without specific amino acids (ActD)
Harvest Cells
Supplement deficient media with radioactive amino
acids
Stimulate Induce Transfect Infect
Detection
Immunoprecipitation
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Pulse-Label Protein Detection
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Radioisotope Methods Pulse-Chase

• Used to detect proteins proteolytically
  processed, degraded, or modified in a given
  amount of time (the chase)
• Cells are pulsed with radioactive substrate
  (e.g., amino acids)
• Cells are chased with excess cold substrate
• Cycloheximide can be added to prevent further
  translation

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Radioisotope Methods Pulse-Chase Basic Protocol
Culture Cells
Stimulate Induce Transfect Infect
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Radioisotope Methods Pulse-Chase Basic Protocol
Starve Cells
Culture Cells
Culture without specific amino acids (ActD)
Stimulate Induce Transfect Infect
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Radioisotope Methods Pulse-Chase Basic Protocol
Starve Cells
Culture Cells
Pulse Label Cells
Culture without specific amino acids (ActD)
Supplement deficient media with radioactive amino
acids
Stimulate Induce Transfect Infect
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Radioisotope Methods Pulse-Chase Basic Protocol
Starve Cells
Chase Cells
Remove radiolabel add excess cold amino
acids (Chx)
Culture Cells
Pulse Label Cells
Culture without specific amino acids (ActD)
Supplement deficient media with radioactive amino
acids
Stimulate Induce Transfect Infect
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Radioisotope Methods Pulse-Chase Basic Protocol
Starve Cells
Chase Cells
Remove radiolabel add excess cold amino
acids (Chx)
Culture Cells
Pulse Label Cells
Culture without specific amino acids (ActD)
Supplement deficient media with radioactive amino
acids
Stimulate Induce Transfect Infect
Harvest Cells
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Radioisotope Methods Pulse-Chase Basic Protocol
Starve Cells
Chase Cells
Remove radiolabel add excess cold amino
acids (Chx)
Culture Cells
Pulse Label Cells
Culture without specific amino acids (ActD)
Supplement deficient media with radioactive amino
acids
Stimulate Induce Transfect Infect
Harvest Cells
Immunoprecipitation
Detection
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Pulse-Chase Protein Detection
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Radioisotope Methods Nucleic Acid Detection

• Nuclear runoff transcription
• Total viral RNA synthesis in infected cells

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Radioisotope Methods Nuclear Runoff Transcription

• Identifies newly-transcribed RNA from isolated
  nuclei
• Measures mostly elongated transcripts that were
  already initiated at time of isolation
• Most sensitive procedure for measuring specific
  gene transcription as a function of cell state

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Reagents for Nuclear Runoff Transcription

• Cultured cells
• Non-SDS lysis buffer (to isolate nuclei)
• Reaction buffer with ATP, GTP, CTP
• a-32P-UTP
• DNase I and Proteinase K
• Materials for RNA extraction
• cDNA of interest immobilized to nitrocellulose
• RNase A

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Nuclear Runoff TranscriptionBasic Protocol
Stimulate Cells
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Nuclear Runoff TranscriptionBasic Protocol
Stimulate Cells
Isolate and Freeze/Thaw Nuclei
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Nuclear Runoff TranscriptionBasic Protocol
Stimulate Cells
Isolate and Freeze/Thaw Nuclei
Add Nucleotides And Perform Transcription
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Nuclear Runoff TranscriptionBasic Protocol
Stimulate Cells
Isolate and Freeze/Thaw Nuclei
Add Nucleotides And Perform Transcription
Stop with DNase and Proteinase K
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Nuclear Runoff TranscriptionBasic Protocol
Stimulate Cells
Isolate and Freeze/Thaw Nuclei
Add Nucleotides And Perform Transcription
Stop with DNase and Proteinase K
Extract RNA
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Nuclear Runoff TranscriptionBasic Protocol
Stimulate Cells
Isolate and Freeze/Thaw Nuclei
Add Nucleotides And Perform Transcription
Stop with DNase and Proteinase K
Extract RNA
Hybridize RNA To cDNA
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Nuclear Runoff TranscriptionBasic Protocol
Stimulate Cells
Isolate and Freeze/Thaw Nuclei
Add Nucleotides And Perform Transcription
Stop with DNase and Proteinase K
Extract RNA
Hybridize RNA To cDNA
Remove Unhybridized RNA with RNase A
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Nuclear Runoff TranscriptionBasic Protocol
Stimulate Cells
Isolate and Freeze/Thaw Nuclei
Add Nucleotides And Perform Transcription
Stop with DNase and Proteinase K
Extract RNA
Hybridize RNA To cDNA
Expose to Film
Remove Unhybridized RNA with RNase A
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Radioisotope Methods Total Viral RNA Synthesis
in Infected Cells

• Viral RNA-dependent RNA polymerases function even
  in the absence of cellular transcription
  (Actinomycin D-resistant)
• Large amounts of RNA produced allow use of
  3H-Uridine for detection

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Reagents for Total Viral RNA Synthesis Assay

• Infected cells
• Cell culture media /- Actinomycin D
• 3H-Uridine
• Non-SDS lysis buffer
• Reagents for TCA precipitation of RNA
• 10 TCA
• 5 TCA
• Glass microfiber filters
• Scintillation fluid, vials, and scintillation
  counter

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Radioisotope Methods Total Viral RNA Synthesis
Culture Cells
Infect
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Radioisotope Methods Total Viral RNA Synthesis
Stop Cellular RNA Synthesis
Culture Cells
Infect
Culture in media ActD
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Radioisotope Methods Total Viral RNA Synthesis
Stop Cellular RNA Synthesis
Culture Cells
Label Cells
Supplement media with radioactive Uridine
Infect
Culture in media ActD
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Radioisotope Methods Total Viral RNA Synthesis
Stop Cellular RNA Synthesis
Culture Cells
Label Cells
Harvest Cells
Supplement media with radioactive Uridine
Infect
Culture in media ActD
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Radioisotope Methods Total Viral RNA Synthesis
Stop Cellular RNA Synthesis
Culture Cells
Label Cells
Harvest Cells
Supplement media with radioactive Uridine
Infect
Culture in media ActD
Detection
TCA Precipitation
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RNA Synthesis Detection3H-Uridine
Incorporation
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Nonradioisotope MethodsBrdU, BrdUTP, and BrUTP
Incorporation

• Brominated deoxyuridine, dUTP and UTP
• BrdU incorporated in cellular DNA in place of
  Thymidine
• Effective label for cell proliferation and cell
  cycle
• BrdUTP incorporated into double-strand breaks of
  DNA by TdT
• Effective label for apoptotic cells
• BrUTP is a substrate for RNA polymerase
• Can monitor transcription, viral RNA synthesis

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Downstream ApplicationsBrdU, BrdUTP, and BrUTP
Detection

• BrdU
• Antibody detection of newly-synthesized DNA
• Fluorescence enhancement or quenching
• BrdUTP
• Antibody detection of newly-synthesized DNA
• TUNEL assay
• BrUTP
• Antibody detection (anti-BrdU) of transcribed RNA

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Nonradioisotope MethodsStable Isotopic Amino
Acids

• SILAC media (Stable Isotopic Labeling of Amino
  acids in Culture) contains light and/or heavy
  isotopes of certain amino acids
• Proteins 100 labeled in 6 passages
• Low background (13C conversion in Arg-Pro
  catalysis)

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Downstream ApplicationsStable Isotopic Amino
Acids

• Proteins detected by mass spectrometry
• Even subtle differential effects on metabolism in
  the presence of drug treatment, siRNA/shRNA
  knockdown, can be analyzed
• More info www.invitrogen.com

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Problem Solving