Exam 3 Protocols

• DNase I Footprinting - to look at the binding of protein(s) to a particular site on DNA
• DNase I Footprinting:

  1. Treat as desired
  2. Collect cells using trypsin
  3. Lyse plasma membrane with NP-40
  4. Spin down and collect nuclei
  5. Lyse nuclear membrane with EDTA
  6. Radioactively label a particular fragment of DNA
  7. Incubate nuclear proteins with labeled DNA
  8. Add DNase I at concentration allowing for 1 cleavage per strand of DNA
  9. Add loading buffer and run DNA on PAGE gel
  10. Expose to film
• DNase I Footprinting loading buffers: Bromophenol Blue, Formaldehyde, and Glycerol
• Formaldehyde causes DNA strands and protein to break apart
• DNase I can't cut where protein is bound
• DNase I Footprinting Advantages: determine if particular DNA sequence is bound by protein
• DNase I Footprinting Disadvantages: Gives no info on the nature of protein bound or if it plays regulatory role (activator, repressor, or both)
• Mobility Shift: Determines whether there are nuclear proteins capable of binding to particular DNA sequence
• Mobility Shift:

  1. Treat cells as desired
  2. Collect cells using trypsin
  3. Lyse plasma membrane with NP-40
  4. Spin down and collect nuclei
  5. Lyse nuclear membrane with EDTA
  6. Radioactively label particular DNA fragment (10-100 bp)
  7. Incubate nuclear proteins with labeled DNA
  8. Add loading buffer and run on PAGE gel
  9. Expose to film
• Mobility shift loading buffers: Glycerol and Bromophenol Blue
• Mobility Shift Advantages: Tell whether proteins are bound to particular DNA fragment; Easy and inexpensive
• Mobility Shift Disadvantages: Can't determine what protein is bound; Only looks at in vitro binding; Doesn't tell if interaction has regulatory role
• Supershift Analysis: Determine whether a specific protein is capable of binding to particular DNA sequence
• Supershift Analysis:

  1. Treat cells as desired
  2. Collect cells using trypsin and Lyse plasma membrane
  3. Spin down and collect nuclei
  4. Lyse nuclear membrane with EDTA
  5. Radioactively label a particular DNA fragment (10-100 bp)
  6. Incubate nuclear proteins with labeled DNA
  7. Add Antibody to protein of interest
  8. Add loading buffer and run on PAGE gel
  9. Expose to film
• Super-Shift Assay Overview:

  • DNA and proteins from nuclear extracts are incubated together
  • Ab to potential proteins are then added to see what is bound to DNA
  • Ab doesn't bind to DNA binding domain of protein
• Supershift Advantages: allows you to determine what particular protein is bound to particular DNA fragment
• Supershift Disadvantages: More expensive than EMSA; Only looks at in vitro binding; Doesn't tell you whether interaction has regulatory role
• Depletion Analysis: Determines whether a specific protein is capable of binding to a particular DNA sequence
• Depletion Analysis:

  1. Treat cells as desired
  2. Collect cells using trypsin and lyse plasma membrane with NP-40
  3. Spin down and collect nuclei
  4. Lyse nuclear membrane with EDTA
  5. Add antibody to protein of interest
  6. Radioactively label a particular DNA fragment (10-100 bp)
  7. Incubate nuclear proteins with labeled DNA
  8. Add loading buffer and run on PAGE gel
  9. Expose to film
• Depletion Assay loading buffers: Glycerol and Bromophenol Blue
• Depletion Assay Advantages: Allows you to determine what particular protein is bound to particular DNA fragment
• Depletion Assay Disadvantages: More expensive than EMSA; Only looks at in vitro binding; Doesn't tell you whether interaction has regulatory role
• Chromatin Immunoprecipitation (ChIP) Assay: To determine if particular protein can bind to specific DNA fragment in vivo
• ChIP Assay:

  1. Treat cells as desired (cells living)
  2. Add formaldehyde to cross-link proteins to DNA (cells dead)
  3. Lyse cells and extract protein/DNA complexes
  4. Digest DNA into fragments using RE
  5. IP with Ab to protein of interest
  6. Add protein K to digest proteins
  7. Isolate DNA from sample
  8. Amplify DNA using primers to DNA sequence of interest
  9. Analyze DNA using Real Time PCR, Agarose gel with ethidium bromide, then DNA Sequencing
• ChIP Assay Advantages: Shows you binding of specific proteins to specific sequence of DNA in vivo
• ChIP Assay Disadvantages: More expensive; More time consuming; Doesn't tell you regulatory role
• Transient Transfection

  1. Create vector with gene that codes for protein of interest
  2. Transfect into cells
  3. Treat one dish of cells and leave others untreated
  4. Co-transfect with another vector that allows you to measure transfection efficiency (vector containing GFP gene commonly used)
• Transient Transfection vector: ORI, Prokaryotic Ab resistance gene, MCS
• Stable Transfection

  1. create vector with ORI, Prokaryotic and Eukaryotic resistance gene, MCS
  2. Add Ab that will kill eukaryotic cells
  3. Grow surviving cells in culture medium
• Prokaryotic Ab resistance gene - selects bacterial cells that have taken up the vector when amplifying plasmids for experiment use
• Eukaryotic Ab resistance gene - selects eukaryotic cells that have taken up the vector to create stably transfected culture of cells
• Stable Transfection - adding Ab to kill eukaryotic cells

  • Only those transfected will survive
  • In a few cells, the vector will integrate into genomic DNA and other vectors will be degraded in a few days
• Stable transfection doesn't need co-transfection as all of the cells have the vector as it was incorporated into genome
• Transient Transfection Advantages: Less expensive, Less time consuming
• Transient Transfection Disadvantages:

  • Must transfect for each experiment (vector degraded rapidly)
  • Have to co-transfect
  • Results less clear (not all cells transfected)
• Stable Transfection Advantages:

  • Transfect once and can use cells for many experiments
  • Don't need to co-transfect
  • Results are more clear (all cells transfected)
• Stable Transfection Disadvantages:

  • More expensive
  • More time consuming
  • Site of integration can affect results or be lethal
• EMSA, ChIP, DNase I footprinting: can determine if a particular protein is bound to the promoter/enhancer region of particular gene of interest
• Promoter Analysis: use of reporter gene to evaluate activity of a promoter/enhancer for particular gene of interest
• Promoter Analysis steps

  1. Create appropriate vector (promoter of interest + reporter gene (Luciferase, CAT))
  2. Transfect (transient or stable) into cells
  3. Treat cells as desired
  4. Measure read-out from reporter gene