Transcription/Translation Protocols

• Northern Blot: Measures steady state levels of mRNA
• Northern Blot:

  1. Treat cells as desired
  2. Extract total RNA from cells and quantitate
  3. Add loading buffer
  4. Boil Samples
  5. Run on agarose gel containing formaldehyde
  6. Stain gel with ethidium bromide
  7. Transfer mRNA from gel to nitrocellulose membrane (if RNA intact)
  8. Hybridize to 32P-cDNA labeled probe specific to mRNA of interest
  9. Autoradiography
• What are the loading buffers used in Northern Blot and mRNA half-life?

  Formaldehyde, Glycerol, Bromophenol blue
• What does formaldehyde do?

  Denatures RNA
• Ethidium bromide shows if mRNA is degraded
• To control for mRNA loading in Northern Blot:

  1. Strip Northern Blot membrane (remove radioactive probe to mRNA B)
  2. Label probe to mRNA that doesn't change in levels
  3. Hybridize to membrane
  4. Expose to film
  5. Densitometry
• What are the Northern Blot loading controls?

  GAPDH, Actin, A-50
• What is loading control is mostly used?

  A-50
• Densitometry: measure size of band in pixels, gene of interest/loading control
• Explanations for Northern Blot

  1. Cell is making more mRNA in response to stimulus (increase transcription)
  2. Cell making same amount of mRNA, but mRNA stays around longer (increased half-life)
• Advantages of Northern Blot?

  Quick, Inexpensive, easy; Specific to particular mRNA; Can be controlled for loading
• Disadvantages of Northern Blot?

  Only looks at steady state levels of RNA
• mRNA half life - determines the half life of mRNA under specific conditions
• mRNA half-life

  1. Treat cells as desired
  2. Add transcriptional inhibitors
  3. Extract total RNA from cells at various times
  4. mRNA concentrations
  5. Add loading buffer
  6. Boil Samples
  7. Run on agarose gel containing formaldehyde
  8. Stain with ethidium bromide (18S and 28S rRNA)
  9. Transfer mRNA from gel to nitrocellulose membrane
  10. Hybridize membrane with radioactive probe complementary to mRNA of interest
  11. Expose membrane to film
  12. Densitometry
• Advantages of mRNA half-life?

  can measure RNA half life
• Disadvantages of mRNA half life?

  Need more dishes, More time consuming
• Nuclear Run-Off - Determines level of transcription taking places at particular time for given gene
• Nuclear Run-Off:

  1. Treat cells as desired
  2. Lyse plasma membrane with NP-40
  3. Spin down and collect nuclei
  4. In vitro transcription reaction
  5. Lyse nuclear membrane with EDTA
  6. Extract mRNA
  7. Immobilize cDNA for mRNA of interest on nitrocellulose
  8. Hybridize cDNA on membrane to radioactive mRNA from samples
  9. Expose membrane to film and perform densitometry
• In a northern the mRNA is not radioactive and the cDNA probe is
• In a nuclear run off the cDNA probe is not radioactive but the mRNA is
• Conclusion of nuclear run off?

  The transcriptional rate ....
• Advantages of nuclear run off?

  Can determine transcriptional rate
• Disadvantages of nuclear run off?

  Harder to perform, No loading control, Time consuming, Need a lot of cells
• In vitro transcription reaction in nuclear run off?

  Add 32P-CTP; UTP, GTP, ATP; RNA Polymerase; Nuclei
• Making Primary Antibody to Vitronectin (mouse ab to human)

  1. Inject mouse with human Vitronectin protein
  2. Mouse will make Ab against this foreign protein
  3. Mouse primary Ab to Vitronectin
• Making Secondary Antibody to Mouse Primary Antibody (goat ab)

  1. Inject goat with mouse primary Ab to Vitronectin
  2. Goat will make Ab against this foreign protein
  3. Typically conjugated to a tag - Fluorescent or Enzyme linked
• A secondary antibody is used because Primary Ab recognize very specific epitopes on antigen and it is difficult to tag without disrupting epitope binding site
• Western Blot: Indicates steady state levels of specific proteins at a given time
• Western Blot steps:

  1. Treat cells as desired (leave some untreated)
  2. Extract protein from cells and quantitate
  3. Add loading buffer
  4. Boil samples
  5. Run on SDS-PAGE gel
  6. Transfer proteins to nitrocellulose membrane
  7. Block membrane in milk or BSA
  8. Add primary Ab to protein of interest and wash
  9. Add secondary Ab conjugated to HRP to primary Ab and wash
  10. Add chemiluminescent reagent that interacts with HRP
  11. Expose to film
• Western Blot protein loading control:

  1. Strip membrane (remove Ab)
  2. Select Ab to a protein that doesn't change levels (ERK-1 / ERK-2)
  3. Hybridize to membrane as done previously
  4. Expose to film
  5. Densitometry
• Western Blot Advantages - Quick and relatively easy; Allows detection of steady state level of specific protein
• Western Blot disadvantages - Only looks at steady state levels of protein; Ab can be expensive / not available
• Western Half Life: determines the half life of specific protein under specific conditions
• Western Half Life steps:

  1. Treat cells as desired
  2. Add translation inhibitors
  3. Extract total protein from cells at various times
  4. Quantitate protein
  5. Add loading buffer
  6. Boil samples
  7. Run on SDS-PAGE gel
  8. Transfer to nitrocellulose membrane
  9. Block membrane in milk or BSA
  10. Add primary Ab and wash
  11. Add secondary Ab conjugated to HRP to primary Ab and wash
  12. Add ECL reagent
  13. Expose to film and densitometry
• Densitometry gives arbitrary values
• Western Half Life doesn't have loading control so have to repeat
• Western Half Life Advantages - Can measure protein half life
• Western Half Life Disadvantages - Need more dishes; More time consuming
• Western Blot and Half Life loading buffers?

  SDS, Beta-ME, Glycerol, Bromophenol Blue
• Western Blot Conclusions:

  1. Cell is making more protein in response to stimulus
  2. Increased half life