In vitrotranslation assay
Translation in vitro is a very powerful technique to follow the protein synthesis and to characterize the product encoded by mRNAs. The cell-free protein synthesizing system faithfully translates the mRNA from exogenous source under optimal conditions. The assay is usually carried out in a small volume of 20-25mL. It could also be scaled-up for preparative purposes.Principle
The cell-free (S-30) extract containing the necessary protein synthesis machinery components translates the genetic message in the mRNA into protein when provided with energy source under proper ionic conditions. The hot TCA precipitable radioactivity due to the labeled. amino acid incorporation is measured.
Materials
» Cell-free Extract {see Preparation of S-30 Extract for Protein Synthesis In vitro)» Salt Mix (10x)
200mM HEPES-KOH (pH 7.6)
750mM Potassium Chloride
25mM Magnesium Acetate
20mM Dithiothreitol
6mM Spermidine (optional)
Store in Aliquots at -20°C
» Energy Mix (5x)
2.5mM ATP (pH 7.0) 25mL 10mM ATP (dipotassium salt)
1.5mM GTP (pH 7.0) 15mL 10mM GTP (trisodium salt)
100mM creatine phosphate (pH 7.0) 10mL 1M creatine phosphate (dipotassium salt)
250mg/mL creatine phosphokinase 25mLcreatine phosphokinase 1mg/mL 25mLH2O
Prepare the 5x mix afresh from stock solution kept at -20°C.
» Amino acid mix
2.4mM each amino acid (19) except the labeled amino acid stored at -20°C.
Procedure
- In an Eppendorf tube mix successively:
- 10mLof 10x salt mix.
- 20mLof 5x energy mix.
- 10mLof amino acid mix.
- 4mL ( > 40 Ci) of 35S methionine ( > 1000 Ci/mmol) or 3H Leucine
- 36mL of S-30 extract (freshly thawn).
- Pipette out into three numbered 0.5mL Eppendorf tubes three different volumes (1, 2 and 4mL) of mRNA (1mg/mL) solution. Normalize the volume to 5mL in each tube by adding sterile distilled water.
- Transfer 20mL of the assay mix (step 1) to each tube containing mRNA.
- To the remaining assay mixture add 5mLof sterile water (control).
- Mix and incubate all the tubes at 25°C for 1h.
- Meanwhile warm 5mM square filter papers (Whatman 3mm) over a hot plate at 70°C.
- After incubation transfer 2mL or 5mL of assay in triplicate onto the filters papers, and dry.
- Precipitate the proteins onto the filters by transferring them to ice-cold 10% trichloro acetic acid (TCA) containing excess unlabeled amino acid for 10 min.
- Boil the filters in 5% TCA for 10 min in a water bath to deacylate the charged tRNAs.
- Wash the filters successively in 5% TCA, ethanol, ethanol-ether mixture and finally ether each step proceeding for 2-3 min.
- Dry the filters at 70°C. Transfer each filter to a scintillation vial, add 2mL scintillation fluid (4g PPO/L toluene) or suitable cocktail and count the radioactivity.
- Include three filters with no solution pipetted onto them during above processing to subtract the background noise.
- Arrest the reaction after incubation (Step 5) by adding 5mLof sample buffer (5x) for SDS-PAGE analysis.
Notes
- S-30 extract 0.3-0.6mL/mL assay can be included.
- At the start of incubation aliquots may be withdrawn and radioactivity counted as a check.
- The energy system may be a failure if there was no incorporation.
- TMV RNA may be programmed in a separate tube to compare the assays.
- Addition of rRNA is beneficial to certain extent although usually not necessary.
Table: The amount of solid ammonium sulphate to be added to a solution to give the desired final saturation at 0°C
Final conc. of amm. sulpahte, % saturation at 0°C |
||||||||||||||||||
| Initial conc. of amm. sulphate | |
20 |
25 |
30 |
35 |
40 |
45 |
50 |
55 |
60 |
65 |
70 |
75 |
80 |
85 |
90 |
95 |
100 |
| g solid amm. sulphate to add | ||||||||||||||||||
0 |
10.7 |
13.6 |
16.6 |
19.7 |
22.9 |
26.2 |
29.5 |
33.1 |
36.6 |
40.4 |
44.2 |
48.3 |
52.3 |
56.7 |
61.1 |
65.9 |
70.7 |
|
5 |
8.0 |
10.9 |
13.9 |
16.8 |
20.0 |
23.2 |
26.6 |
30.0 |
33.6 |
37.3 |
41.1 |
45.0 |
49.1 |
53.3 |
57.8 |
62.4 |
67.1 |
|
10 |
5.4 |
8.2 |
11.1 |
14.1 |
17.1 |
20.3 |
23.6 |
27.0 |
30.5 |
34.2 |
37.9 |
41.8 |
45.8 |
50.0 |
54.5 |
58.9 |
63.6 |
|
15 |
2.6 |
5.5 |
8.3 |
11.3 |
14.3 |
17.4 |
20.7 |
24.0 |
27.5 |
31.0 |
34.8 |
38.6 |
42.6 |
46.6 |
51.0 |
55.5 |
60.0 |
|
20 |
0 |
2.7 |
5.6 |
8.4 |
11.5 |
14.5 |
17.7 |
21.0 |
24.4 |
28.0 |
31.6 |
35.4 |
39.2 |
43.3 |
47.6 |
51.9 |
56.5 |
|
25 |
|
0 |
2.7 |
5.7 |
8.5 |
11.7 |
14.8 |
18.2 |
21.4 |
24.8 |
28.4 |
32.1 |
36.0 |
40.1 |
44.2 |
48.5 |
52.9 |
|
30 |
|
|
0 |
2.8 |
5.7 |
8.7 |
11.9 |
15.0 |
18.4 |
21.7 |
25.3 |
28.9 |
32.8 |
36.7 |
40.8 |
45.1 |
49.5 |
|
35 |
|
|
|
0 |
2.8 |
5.8 |
8.8 |
12.0 |
15.3 |
18.7 |
22.1 |
25.8 |
29.5 |
33.4 |
37.4 |
41.6 |
45.9 |
|
40 |
|
|
|
|
0 |
2.9 |
5.9 |
9.0 |
12.2 |
15.5 |
19.0 |
22.5 |
26.2 |
30.0 |
34.0 |
38.1 |
42.4 |
|
45 |
|
|
|
|
|
0 |
2.9 |
6.0 |
9.1 |
12.5 |
15.8 |
19.3 |
22.9 |
26.7 |
30.6 |
34.7 |
38.8 |
|
50 |
|
|
|
|
|
|
0 |
3.0 |
6.1 |
9.3 |
12.7 |
16.1 |
19.7 |
23.3 |
27.2 |
31.2 |
35.3 |
|
55 |
|
|
|
|
|
|
|
0 |
3.0 |
6.2 |
9.4 |
12.9 |
16.3 |
20.0 |
23.8 |
27.7 |
31.7 |
|
60 |
|
|
|
|
|
|
|
|
0 |
3.1 |
6.3 |
9.6 |
13.1 |
16.6 |
20.4 |
24.2 |
28.3 |
|
65 |
|
|
|
|
|
|
|
|
|
0 |
3.1 |
6.4 |
9.8 |
13.4 |
17.0 |
20.8 |
24.7 |
|
70 |
|
|
|
|
|
|
|
|
|
|
0 |
3.2 |
6.6 |
10.0 |
13.6 |
17.3 |
21.2 |
|
75 |
|
|
|
|
|
|
|
|
|
|
|
0 |
3.2 |
6.7 |
10.2 |
13.9 |
17.6 |
|
80 |
|
|
|
|
|
|
|
|
|
|
|
|
0 |
3.3 |
6.8 |
10.4 |
14.1 |
|
85 |
|
|
|
|
|
|
|
|
|
|
|
|
|
0 |
3.4 |
6.9 |
10.6 |
|
90 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
0 |
3.4 |
7.1 |
|
95 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
0 |
3.5 |
|
100 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
0 |
|
References
1. Carlier, A R, Manickam, A and Peumans, W J (1980) Planta 149 227.2. Marcus, A, Efron, D and Weeks, D P (1974) Meth Enzymol 30 749.
3. Roberts, B E and Paterson, A M (1973) Proc NatlAcad Sci USA 70 2330.
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