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  Section: Biotechnology Methods » Molecular Biology
 
 
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Protein Synthesis in Cell Free Systems

 
     
 
Content
Molecular Biology
  The Central Dogma
  Protein Synthesis in Cell Free Systems
  Chromosomes
  Polytene Chromosomes of Dipterans
  Salivary Gland Preparation (Squash Technique)
  Extraction of Chromatin
  Chromatin Electrophoresis
  Extraction and Electrophoresis of Histones
  Karyotype Analysis
  In Situ Hybridization
  Culturing Peripheral Blood Lymphocytes
  Microslide Preparation of Metaphases for In-Situ Hybridization
  Staining Chromosomes (G-Banding)
  Nucleic Acids
  Extraction of DNA from Bovine Spleen
  Purification of DNA
  Characterization of DNA
  DNA-Dische Diphenylamine Determination
  Melting Point Determination
  CsCl-Density Separation of DNA
  Phenol Extraction of rRNA (Rat liver)
  Spectrophotometric Analysis of rRNA
  Determination of Amount of RNA by the Orcinol Method
  Sucrose Density Fractionation
  Nucleotide Composition of RNA
  Isolation of Genomic DNA—DNA Extraction Procedure
  Isolation of Genomic DNA from Bacterial Cells
  Preparation of Genomic DNA from Bacteria
  Extraction of Genomic DNA from Plant Source
  Extraction of DNA from Goat Liver
  Isolation of Cotton Genomic DNA from Leaf Tissue
  Arabidopsis Thaliana DNA Isolation
  Plant DNA Extraction
  Phenol/Chloroform Extraction of DNA
  Ethanol Precipitation of DNA
  Isolation of Mitochondrial DNA
  Isolation of Chloroplast DNA
  DNA Extraction of Rhizobium (CsCl Method)
  Isolation of Plasmids
  RNA Isolation
  Preparation of Vanadyl-Ribonucleoside Complexes that Inhibit Ribonuclease Activity
  RNA Extraction Method for Cotton
  Isolation of RNA from Bacteroids
  Isolation of RNA from Free-Living Rhizobia
  Estimation of DNA purity and Quantification
  Fungal DNA Isolation
  Methylene Blue DNA Staining
  Transformation
  Blotting Techniques—Southern, Northern, Western Blotting
  Preparing the Probe
  Southern Blotting (First Method)
  Southern Blotting (Second Method)
  Western Blotting
  Western Blot Analysis of Epitoped-tagged Proteins using the Chemifluorescent Detection Method for Alkaline Phosphatase-conjugated Antibodies
  Southern Blot
  Southern Analysis of Mouse Toe/Tail DNA
  Northern Blotting
  Restriction Digestion Methods—Restriction Enzyme Digests
  Restriction Digestion of Plasmid, Cosmid, and Phage DNAs
  Manual Method of Restriction Digestion of Human DNA
  Preparation of High-Molecular-Weight Human DNA Restriction Fragments in Agarose Plugs
  Restriction Enzyme Digestion of DNA
  Electroelution of DNA Fragments from Agarose into Dialysis Tubing
  Isolation of Restriction Fragments from Agarose Gels by Collection onto DEAE Cellulose
  Ligation of Insert DNA to Vector DNA
  PCR Methods (Polymerase Chain Reaction)
  Polymerase Chain Reaction
  DNA Amplification by the PCR Method

Materials
  • Suspension culture of fibroblast cells (1 liter)\
  • 35 mM of Tris-HCl, pH 7.4, 140 mM NaCl (TBS buffer)
  • 10 mM of Tris-HCl, pH 7.5, 10 mM KCl, and 1.5 mM magnesium acetate (TBS-M)
  • 10X TBS-M: 200 mM of Tris-HCl, pH 7.5, 1200 mM KCl, 50 mM magnesium
  • acetate and 70 mM β--mercaptoethanol
  • 10X solution of 20 amino acids
  • Teflon homogenizer
  • Refrigerated preparative centrifuge
  • Saturated (NH4)2SO4
  • TBS-M plus 20% (v/v) glycerol
  • 1X TBS-M buffer containing 1.0 M sucrose
  • Sephadex G-25 column equilibrated with 1X TBS-M buffer
  • Liquid nitrogen storage
  • Reaction mixture for protein synthesis, containing the following in a total volume of 50 µL

    Tris-HCl, pH 7.5
    1.5 µm
    Mg acetate
    0.15-0.20 µm
    KCl 4.0-5.0 µm
    β--Mercaptoethanol 0.25 µm
    ATP 0.05 µm
    GTP 0.005 µm
    Creatine phosphate 0.50 µm
    Creatine kinase 8.0 µg
    Each of 19 amino acids(-leucine)
    2.0 nmol
    14C-leucine (150 µCi/mmol)
    0.125 µCi
    Ribosome fraction
    1 to 2 A260 units
    Viral mRNA or Globin 9S mRNA 2.0 to 5.0 µg
    or
    Poly U 10.0 µg
Procedure
  1. Chill the suspension culture (~109 cells) rapidly in an ice bath. Collect the cells as a pellet by centrifugation at 600 xg for 10 minutes at 4°C. Resuspend the cells in TBS buffer and wash them 3 times with cold TBS buffer.
  2. Suspend the final pellet in 2 volumes of TBS-M for 5 minutes at 0°C and homogenize the cells with 10 to 20 strokes in a tight-fitting Teflon homogenizer.
  3. For each 0.9 mL of homogenate, add 0.1 mL of concentrated 10X TBS-M buffer. Centrifuge the mixture at 10000 xg for 10 minutes at 4°C.
  4. Decant and collect the supernatant extract and adjust the extract such that the following are added to yield final concentrations:
    • ATP to 1.0 mM ATP
    • GTP to 0.1 mM GTP
    • Creatine phosphate to 10 mM
    • Creatine kinase to 160 µg/mL
    • Amino acids to 40 µm each.
  5. Incubate the mixture for 45 minutes at 37°C.
  6. Centrifuge the mixture at 10000 xg for 10 minutes at room temperature. Cool the supernatant and pass it through a Sephadex G-25 column at 4°C.
  7. Turn on a UV spectrophotometer and adjust the wavelength to 260 nm. Blank the instrument with TBS buffer.
  8. Centrifuge the filtrate excluded from the Sephadex column at 165,000 xg for 90 minutes at 4°C.
  9. Precipitate the proteins within the supernatant by the addition of saturated (NH4)2SO4 to yield a final 60% (NH4)2SO4. Collect the precipitate by centrifugation.
  10. Dissolve the precipitate in TBS-M buffer and dialyze it against the same buffer containing glycerol.
  11. Suspend the resulting ribosome pellet in 1X TBS-M buffer containing 0.25 M sucrose. Place 5 mL of TBS buffer with 1 M sucrose into the bottom of a centrifuge tube and layer the suspended ribosomes on top. Centrifuge at 216000 xg for 2.5 hours at 4°C.
  12. Wash the resulting pellet with TBS-M buffer, and resuspend it in the same buffer with 0.25 M sucrose.
  13. Determine the ribosome concentration using a UV spectrophotometer to measure the A260. The extinction coefficient for ribosomes is 12 A units per mg per mL at 260 nm.
  14. The ribosomes may be frozen and stored in liquid nitrogen, or used for in vitro protein synthesis. If frozen, they should be thawed only once prior to use. To test for protein synthesis, prepare the reaction mixture for protein synthesis.
  15. Incubate the reaction mixture at 37°C for 60 minutes. Terminate the reaction by pipetting 40 µL of the mixture onto a 2.5-cm disk of Whatman 3-MM filter paper. Dip the disk into cold 10% TCA for 15 minutes and then in 5% TCA at 90°C for 15 minutes.
  16. Rinse the disk twice in 5% TCA for 5 minutes, once in alcohol:ether (1:1), and then dry it.
  17. Place the disk into a scintillation vial and add a toluene-based fluor.
  18. Measure the amount of radioactively labeled amino acid incorporated into protein.
  19. Graph the protein synthesized versus time.
Optional
For advanced work, compare the activity of ribosomes isolated from the fibroblast cultures to those isolated from a prokaryote culture, a plant (yeast or pea seedlings), and from genetic mutants known to alter the structure of either rRNA or any of the ribosome structural proteins.

 
     
 
 
     




     
 
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