Protocols and Applications Guide (US Letter Size) - Promega

||||| 5Protein Expression
• radiolabeled amino acid
• Nuclease-Free Water (Cat.# P1193)
1. Mix the following components on ice, in the order
given, in a sterile 1.5ml microcentrifuge tube.
Translation Reaction with TNT® Rabbit Reticulocyte
Components
TNT® Lysate
TNT® Reaction Buffer
Amino Acid Mixture Minus
Methionine, 1mM
TNT® RNA Polymerase (SP6, T3 or T7)
[35S]methionine (1,200Ci/mmol, at
10mCi/ml)
Nuclease-Free Water
Plasmid DNA, 0.5mg
Canine Microsomal Membranes
final volume
Volume
12.5µl
0.5µl
0.5µl
0.5µl
2.0µl
5.5µl
0.5µl
2.5µl
25.0µl
Translation Reaction with Rabbit Reticulocyte Lysate
System, Nuclease-Treated
Components
Volume
Rabbit Reticulocyte Lysate,
17.5µl
Nuclease-Treated
1mM Amino Acid Mixture (Minus
0.5µl
Methionine)
[35S]methionine (1,200Ci/mmol, at
2.0µl
10mCi/ml)
Nuclease-Free Water
2.2µl
Canine Microsomal Membranes
1.8µl
RNA substrate in water 1.0µl
1
final volume
25.0µl
1 For the control reactions, use pre β-lactamase and α-factor mRNA
at 0.1µg/ml.
2. Incubate for 90 minutes at 30°C.
3. Analyze results.
C. Analysis of Results
When using 1.8µl of Microsomal Membranes per 25µl of
translation mix, 90% of pre-β-lactamase will be processed
to β-lactamase. The same amount of membranes will
process 75–90% of α-factor to core glycosylated forms of
α-factor. Upon SDS-PAGE, the precursor for β-lactamase
migrates at 31.5kDa and the processed β-lactamase at
28.9kDa. The precursor for the α-factor migrates at 18.6kDa,
and the core-glycosylated α-factor has a molecular weight
of 32.0kDa but will migrate faster than the β-lactamase
precursor (Figure 5.4).
In some cases, it is difficult to determine if efficient
processing or glycosylation has occurred by gel analysis
alone. These alternative assays for detecting co-translational
processing events may be useful. A general assay for
co-translational processing uses the protection afforded the
translocated protein domain by the lipid bilayer of the
Protocols & Applications Guide
www.promega.com
rev. 6/09
microsomal membrane. In this assay, protein domains are
judged to be translocated if they are observed to be
protected from exogenously added protease. To confirm
that protection is due to the lipid bilayer, addition of 0.1%
non-ionic detergent (such as Triton® X-100 or Nikkol)
solubilizes the membrane and restores susceptibility to
protease. Many proteases have proven useful for
monitoring translocation in this fashion including protease
K and trypsin (final concentration 0.1mg/ml; Gross et al.
1988).
An alternative procedure uses endoglycosidase H to
determine the extent of glycosylation of translation products
(Andrews, 1987). In cell-free systems, N-linked
glycosylation occurs only within intact microsomes.
Endoglycosidase H cleaves the internal
N-acetylglucosamine residues of high mannose
carbohydrates, resulting in a shift in apparent molecular
weight on SDS-polyacrylamide gels to a position very close
to that of the nonglycosylated species. The reaction
conditions (0.1% SDS, 0.1M sodium citrate [pH 5.5]
incubation at 37°C for 12 hours) are not compatible with
those required to maintain membrane integrity. For this
reason, translocated polypeptides are not “protected” from
digestion with endoglycosidase H.
Additional Resources for Canine Microsomal Membranes
Technical Bulletins and Manuals
TM231 Canine Pancreatic Microsomal Membranes
Technical Manual
(www.promega.com
/tbs/tm231/tm231.html)
Promega Publications
PN038 Post-translational processing: Use of the
TNT® Lysate Systems with Canine
Microsomal Membranes
(www.promega.com
/pnotes/38/38_15/38_15.htm)
PN070 Applications of Promega's In Vitro
Expression Systems
(www.promega.com
/pnotes/70/7618_02/7618_02.html)
Citations
He, W. et al. (2007) The membrane topology of RTN3 and
its effect on binding of RTN3 to BACE1. J. Biol. Chem. 282,
29144-29151.
The authors of this study determined the membrane
topology of reticulon 3 (RTN3), an integral membrane
protein that is expressed at high levels in neruons and has
been show to negatively regulate the activity of BACE1
(Beta site APP-Cleaving Enzyme). Disruption of RTN3 is
associated with incidence of dystrophic neurites in AD
brain. RTN3 was translated using the TNT® Quick Coupled
Transcription/Translation System in the presence of Canine
Microsomal Membranes and labeled using the Transcend
Non-Radioactive Translation Detection System.
PubMed Number: 17699523
PROTOCOLS & APPLICATIONS GUIDE 5-13