molecular biology techniques - study of...
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Molecular Biology Techniques USABO
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Basics
• PCR
• Sanger method for sequencing
•Microscopy techniques
Light
Fluorescence
XFP tagging, bleaching, toxicity
Electron
(Dis)advantages of each type
Basic preparation steps
• Northern, Southern, Western blots
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Protein Gel Electrophoresis/Western
Western Blot
• Phosphate-state specific antibodies
•Monoclonal vs. polyclonal
SDS-PAGE Transfer to membrane
Immunoblotting
• Native vs. denaturing (SDS-PAGE)
• Specialized: 2D gels, pH gradients (pI)
• Phosphate-state specific antibodies
•Monoclonal vs. polyclonal
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Protein-Protein Interactions: Two-Hybrid
Bait fusion: DNA binding
protein + bait protein
Target fusion: RNA polymerase
+ target protein
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Protein-Protein Interactions: Two-
Hybrid • Bait fusion protein
binds sequence upstream of reporter and recruits polymerase iff bait and target interact
• Use with screens
• Usually in yeast
• Sometimes in bacteria
•What are the (dis)advantages of each?
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Protein-Protein Interactions: EMSA
• Electrophoretic mobility shift
assay
•What if there are multiple
shifts?
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Protein-Protein Interactions: FRET
• Fluorescence resonance energy transfer
Two proteins, each fused to a different XFP
variant
Use laser @ excitation wavelength of one fusion
Examine emission wavelength of other fusion
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Protein-Protein Interactions
2-hybrid DNA sequences Protein interactions, in
model systems
EMSA Proteins, often
from DNA sequences
Biochemical interactions, in
vitro, identity not guaranteed
Mass spec if from cell lysates
FRET Suspected interacting
proteins, DNA sequences must be known
Interactions, in vitro
Affinity Chromatography
Proteins
Biochemical interactions, in
vitro, identity not guaranteed
Mass spec
X-ray crystallography
Purified protein(s)
Make crystals Identify binding partners
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DNA Cloning
• Restriction enzyme use requires sequence knowledge
• Plasmids designed for cloning often have restriction sites that occur uniquely on plasmid for inserting several fragments in succession
• Retroviruses can allow for genomic integration> cannot replicate as rapidly as a high copy number plasmid
• Introduction of DNA: electroporation, calcium chloride, liposomes, viral constructs
• Replica plating
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DNA extraction/miniprep
• Break cell membranes/wall • Remove organelles /fragments • Remove proteins, RNA, genomic DNA (if isolating
plasmid)
Prep
Overnight culture (7+
hrs)
Pellet
Resuspend
Lyse
Alkali lysis (NaOH/ SDS)
Lysozyme
Precipitate genomic
DNA
KC2H3O2 (also for
neutralizing NaOH)
Keep supernatan
t
Remove RNA
Isopropanol to precipitate nucleic acids
Pellet
RNAse treatment
Remove proteins
Phenol/ chloroform/ isoamyl alcohol to denature
proteins
Centrifuge organic phase to bottom
Proteins @ bottom/interface
Keep supernatant
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DNA gel electrophoresis
• Agarose vs polyacrylamide gels
• Circular vs linear
• Supercoiling and branching
• Footprinting (DNAse I)
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Analysis of Function
• RNAi
siRNA effect (exact match) mRNA degradation
miRNA (close match) transcription and
translation block
Inject RNA, soak in RNA, viral vector, expression
of shRNA
• Antibodies may knock down some protein
activity
• Complementation analysis
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pho4
pho80
pho81
Phosphatase secretion
Deduce the Phosphatase Secretion
Pathway
Which is downstream?
pho80 or pho81?
pho4 or pho80?