Protocols

• Protein Protocols

  Chromatography (Ion Exchange, Gel Filtration, Affinity), Tagged proteins, SDS-PAGE, 2-D Gel Electrophoresis
• Chromatography steps:

  1. Prepare sample as desired
  2. Prepare/obtain column with matrix
  3. Run sample over column
  4. Add solvent to elute flow through
  5. Elute desired bound molecules from column
• SDS-PAGE steps:

  1. Treat cells as desired (may/may not add 35-S methionine)
  2. Extract proteins
  3. Protein concentrations
  4. Add sample buffer
  5. Boil samples
  6. Run on SDS-PAGE gel
  7. Detect proteins
• SDS PAGE buffers

  SDS, Glycerol, Bromophenol Blue, Beta-mercaptoethanol
• SDS buffer denatures, negates intrinsic charge, and solubilized proteins
• Glycerol buffer makes samples thicker
• Bromophenol blue shows where gel is
• Beta-mercaptoethanol breaks disulfide bonds
• Autoradiography is only used to detect proteins if 35-S methionine is used
• Explanations for SDS PAGE and 2D gel electrophoresis

  More of singular protein, More types of proteins, Half-life increased, Gel loaded more in one lane
• Conclusion for SDS-PAGE

  Steady state level ....
• 2D Gel Electrophoresis steps:

  1. Treat cells as desired
  2. Extract proteins
  3. Protein concentrations
  4. Add sample buffer
  5. Run samples on pH gradient IEF strip after which electrical field is applied
  6. Lay IEF strip on top of SDS-PAGE and run gel
  7. Detect proteins
• 2D gel buffers

  Urea, non-ionic detergent, Beta-ME, glycerol, bromophenol blue
• Detecting proteins is done by autoradiography or Coomassie Blue, Gold/Silver Stains
• What does Non-ionic detergent do in 2D Gel?

  solubilizes proteins
• What does Urea do in 2D Gel Electrophoresis?

  Denatures (not disulfide bonds)
• Ion-exchange chromatography matrices

  DEAE-Dextran (+), CM-Cellulose (-), Phosphocellulose (-)
• Gel-Filtration chromatography matrices

  Dextran, Agarose, Acrylamide
• Affinity chromatography matrices

  Substrates, Enzymes, Antibodies
• Ion Exchange - change in pH
• Gel Filtration - Size exclusion (time)
• Affinity chromatography - excess substrate, change in salt, change in pH
• Creating Genomic Library Steps:

  1. Extract total genomic DNA
  2. Digest DNA with REs to create sticky ends
  3. Cut vector with same RE
  4. Ligate DNA fragments into vector
  5. Make bacteria competent using CaCl2
  6. Transform into bacteria
  7. Grow on agar plates with Ab
• Make bacteria competent and transform bacteria:

  1. Place bacterial cells on ice with CaCl2
  2. Add vectors
  3. Transform vectors into bacteria
  4. Heat shock and recover
• Colony Lift:

  1. Spot bacteria onto nitrocellulose membrane
  2. Incubate membrane with solution to lyse bacteria, denature DNA, and adhere DNA
  3. Add radioactive probe complimentary to gene of interest
  4. Expose membrane to film
  5. Match spot on film to original bacterial colony
• Isolate DNA fragments from vector: (last steps for genomic and cDNA)

  1. Grow colonies in nutrient broth overnight to replicate
  2. Lyse bacteria and spin to pellet large cell components
  3. Collect Supernatant
  4. Add EtOH to precipitate plasmid DNA
  5. Centrifuge to pellet plasmid DNA
  6. Resuspend pellet
  7. Add RE as used to put fragment in vector
  8. Run on agarose gel
  9. Isolate DNA fragments from gel
  10. PCR and Sequence DNA
• Creating cDNA Library steps:

  1. Treat cells as desired
  2. Extract total mRNA
  3. Add poly-T primer to poly-A tail of extracted mRNA
  4. Add Reverse transcriptase
  5. Add RNase H
  6. Add poly-A primer to poly-T tail on cDNA
  7. Add DNA polymerase
  8. Cut vector with RE to create blunt ends
  9. Ligate cDNA into vectors
  10. Make bacteria competent with CaCl2
  11. Transform into bacteria
  12. Grow on agar plates containing Ab
• DNA sequencing steps:

  1. Obtain fragment of interest
  2. Denature DNA strands
  3. Set up 4 tubes containing denatured DNA, dNTPs, radioactive primers, and DNA polymerase
  4. Add one of each ddNTPs to each of 4 tubes
  5. Incubate to allow reaction
  6. Run samples on PAGE gel
  7. Expose to film
  8. Read DNA sequence
• Sodium Bisulfite steps:

  1. Treat cells as desired
  2. Extract DNA from cells
  3. Digest DNA with RE
  4. Treat DNA fragments with Sodium Bisulfite
  5. Amplify using PCR with specific primers
  6. Sequence DNA