Protocols and Applications Guide (US Letter Size) - Promega

||||| 5Protein Expression
mRNAs commonly exhibit different optimum salt
concentrations for translation. Furthermore, small variations
in salt concentration can lead to dramatic differences in
translation efficiency. The Flexi® Rabbit Reticulocyte Lysate
System allows optimization of a wide range of parameters,
including Mg2+ and K+ concentrations, and offers the choice
of adding DTT. To help optimize Mg2+ for a specific
message, the endogenous Mg2+ concentration of each lysate
batch is stated on the product insert. The Flexi® System
also offers the choice of three amino acid mixtures and
includes a control RNA encoding the firefly luciferase gene.
For a detailed protocol and background information about
this system, please see Technical Bulletin #TB127
(www.promega.com/tbs/tb127/tb127.html).
Protocol
Materials Required:
• Flexi® Rabbit Reticulocyte Lysate System (Cat.# L4540)
• RNasin® Ribonuclease Inhibitor or RNasin® Plus RNase
Inhibitor (Cat.# N2111 or N2611)
• Nuclease-Free Water (Cat.# P1193)
• radiolabeled amino acid (for radioactive detection) or
Transcend tRNA (Cat.# L5061) or Transcend
Colorimetric (Cat.# L5070) or Chemiluminescent (Cat.#
L5080) Translation Detection System (for
non-radioactive detection) or FluoroTect GreenLys in
vitro Translation Labeling System (for fluorescent
detection; Cat.# L5001)
The following is a general guideline for setting up a Flexi®
Lysate translation reaction. Also provided is an example
of a standard reaction. The reaction uses [35S]methionine
as the radiolabel; other isotopes may also be used (see Table
5.3). For the positive control reaction, use 1–2µl of the
Luciferase Control RNA supplied.
1. Assemble the following reaction components in a 0.5ml
or 1.5ml tube.
Note: We recommend also including a negative control
reaction containing no added template to allow
measurement of background incorporation of labeled
amino acids.
Component
Flexi® Rabbit Reticulocyte Lysate
Amino Acid Mixture Minus
Methionine, 1mM
[35S]methionine (1,200Ci/mmol at
10mCi/ml)
Magnesium Acetate, 25mM
Potassium Chloride, 2.5M
DTT, 100mM
RNasin® Ribonuclease Inhibitor
(40u/ml)
RNA substrate
Nuclease-Free Water to final volume
Protocols & Applications Guide
www.promega.com
rev. 6/09
Volume
33µl
1µl
2µl
0–4µl
1.4µl
0–1µl
1µl
1–12µl
50µl
2. Incubate the translation reaction at 30°C for 60–90
minutes.
3. Analyze the results of translation.
C. RNA Template Considerations
Use a final concentration of 5–80µg/ml of in vitro transcripts
produced with the RiboMAX Large Scale RNA
Production Systems (Cat.# P1280 and P1300) for the
translation. RNA from other standard transcription
procedures may contain components at concentrations that
inhibit translation. Therefore, a lower concentration,
5–20µg/ml of in vitro transcript, should be used with these
systems. The optimal RNA concentration should be
determined before performing experiments. Average
preparations of mRNA stimulate translation about 10- to
20-fold over background (i.e., no exogenous RNA template).
To determine the optimal concentration, serially dilute your
RNA template first and then add the same volume of RNA
to each reaction. This ensures that other variables are kept
constant. In addition, the presence of certain nucleic acid
sequence elements can have profound effects on initiation
fidelity and translation efficiency; 3´-poly(A)+ sequences,
5´-caps, 5´-untranslated regions and the sequence context
around the AUG start, or secondary AUGs in the sequence
(Kozak, 1990).
The presence of inhibitors can significantly reduce
translation efficiency. Oxidized thiols, low concentrations
of double-stranded RNA and polysaccharides are typical
inhibitors of translation in rabbit reticulocyte lysate (Jackson
and Hunt, 1983). To determine if inhibitors are present in
your mRNA preparation, mix your RNA with Luciferase
Control RNA and determine if translation of luciferase
RNA is inhibited relative to a control translation containing
only the luciferase RNA. Residual ethanol should also be
removed from mRNA preparations and labeled amino
acids before they are added to the translation reaction.
You may need to optimize the potassium and magnesium
concentrations in your translation reactions. Addition of
0.5–2.5mM Mg2+ is generally sufficient for the majority of
mRNAs. See Tables 5.4 and 5.5 for the concentrations of
key components present in the lysate.
Table 5.4. Final Concentrations of Rabbit Reticulocyte
Lysate Components in a 50µl Translation Reaction.
Final
Components
Concentration
Creatine phosphate
10mM
Creatine phosphokinase
50µg/ml
DTT
2mM
Calf liver tRNA
50µg/ml
Potassium acetate
79mM
Magnesium acetate
0.5mM
Hemin
0.02mM
PROTOCOLS & APPLICATIONS GUIDE 5-9