Exercise 15.1 - Protein Syntheses in Cell Free Systems
- Suspension culture of fibroblast cells (1 liter)
- 35 mM Tris-HCl, pH 7.4, 140 mM NaCl (TBS buffer)
- 10 mM Tris-HCl, pH 7.5, 10 mM KCl, and 1.5 mM magnesium acetate (TBS-M)
- 10X TBS-M
200 mM Tris-HCl, pH 7.5, 1200 mM KCl, 50 mM magnesium acetate
and 70 mM -mercaptoethanol
- 10X solution of 20 amino acids
- Teflon homogenizer
- Refrigerated preparative centrifuge
- Sat. (NH)SO
- TBS-M plus 20% (v/v) glycerol
- 1X TBS-M buffer containing 0.25 M sucrose
- 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
||0.15 - 0.20 µm
||4.0 - 5.0 µm
| Creatine kinase
| Each of 19 amino acids(-leucine)
|| 2.0 nmol
|C-leucine (150 mCi/mmol)
||1 to 2 A units
| Viral mRNA or Globin 9S mRNA
||2.0 to 5.0 µg
- Chill the suspension culture (app.
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 three times with cold TBS buffer.
- Suspend the final pellet in two 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.
- For each 0.9 ml of homogenate, add 0.1 ml of concentrated
10X TBS-M buffer. Centrifuge the mixture at 10,000 xg for 10
minutes at 4° C.
- Decant and collect the supernatant extract and adjust the
extract such that the following are added to yield final
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
- Incubate the mixture for 45 minutes at 37° C.
- Centrifuge the mixture at 10,000 xg for 10 minutes at room
temperature. Cool the supernatant and pass the supernatant
through a Sephadex G-25 column at 4° C.
- Turn on a UV spectrophotometer and adjust the wavelength to
260 nm. Blank the instrument with TBS buffer.
- Centrifuge the filtrate excluded from the Sephadex column at
165,000 xg for 90 minutes at 4° C.
- Precipitate the proteins within the supernatant by the
addition of saturated (NH)SO to yield a final 60% (NH)SO. Collect the precipitate by
- Dissolve the precipitate in TBS-M buffer and dialyze it
against the same buffer containing glycerol.
- Suspend the resulting ribosome pellet in 1X TBS-M buffer
containing 0.25 M sucrose. Place 5 ml of TBS buffer with 1.0 M
sucrose into the bottom of a centrifuge tube and layer the
suspended ribosomes on top. Centrifuge at 216,000 xg for 2.5
hours at 4° C.
- Wash the resulting pellet with TBS-M buffer, and resuspend
it in the same buffer with 0.25 M sucrose.
- Determine the ribosome concentration using a UV
spectrophotometer to measure the A. The
extinction coefficient for ribosomes is 12 A units per mg per ml
at 260 nm.
- 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
- 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 3MM filter paper. Dip the
disk into cold 10% TCA for 15 minutes and then in 5% TCA at
90° C for 15 minutes.
- Rinse the disk twice in 5% TCA for 5 minutes, once in
alcohol:ether (1:1), and then dry it.
- Place the disk into a scintillation vial and add a
toluene-based fluor. Refer to Appendix H for details on the use of a
- Measure the amount of radioactively labeled amino acid
incorporated into protein.
- Graph the protein synthesized versus time.
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 µ tants known to alter the structure of either rRNA or
any of the ribosome structural proteins.
If sources of mRNA are available (or if the time is taken to
extract RNA and electrophoretically purify it), then comparisons
of the rate of protein synthesis can be made within this system.
Finally, once the proteins are synthesized, they can be separated
on SDS-PAGE and rates of synthesis can be determined by both time
of incorporation of leucine and by increasing molecular weights.
The latter will also demonstrate the possible presence of
µ ltiple RNA's with the system, whereas the radioactivity will
mask the presence of heterogenous RNA.
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Cell Biology Laboratory Manual
Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,
St. Peter, MN 56082 -- firstname.lastname@example.org